Nucleotide sequences of canola and soybean palmitoyl-ACP thioesterase genes and their use in the regulation of fatty acid content of the oils of soybean and canola plants

ABSTRACT

The preparation and use of nucleic acid fragments encoding acyl-acyl carrier protein thioesterase enzymes to modify plant lipid composition are disclosed. Also disclosed are chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences may be used to create transgenic plants with altered levels of saturated fatty acids.

This application is a 371 of PCT/US95/10627 filed Aug. 25, 1995 which isa continuation of U.S. application Ser. No. 08/299,044, filed Aug. 31,1994, now abandoned.

FIELD OF INVENTION

The invention relates to the preparation and use of nucleic acidfragments encoding acyl-acyl carrier protein thioesterase enzymes tomodify plant lipid composition. Chimeric genes incorporating suchnucleic acid fragments and suitable regulatory sequences may be used tocreate transgenic plants with altered levels of saturated fatty acids.

BACKGROUND OF THE INVENTION

Plant lipids have a variety of industrial and nutritional uses and arecentral to plant membrane function and climatic adaptation. These lipidsrepresent a vast array of chemical structures, and these structuresdetermine the physiological and industrial properties of the lipid. Manyof these structures result either directly or indirectly from metabolicprocesses that alter the degree of saturation of the lipid.

Plant lipids find their major use as edible oils in the form oftriacylglycerols. The specific performance and health attributes ofedible oils are determined largely by their fatty acid composition. Mostvegetable oils derived from commercial plant varieties are composedprimarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic(18:2) and linolenic (18:3) acids. Palmitic and stearic acids are,respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic,linoleic, and linolenic acids are 18-carbon-long, unsaturated fattyacids containing one, two, and three double bonds, respectively. Oleicacid is referred to as a mono-unsaturated fatty acid, while linoleic andlinolenic acids are referred to as poly-unsaturated fatty acids. Therelative amounts of saturated and unsaturated fatty acids in commonlyused, edible vegetable oils are summarized below (Table 1):

                  TABLE 1    ______________________________________    Percentages of Saturated and Unsaturated Fatty    Acids in the Oils of Selected Oil Crops             Saturated  Mono-unsaturated                                    Poly-unsaturated    ______________________________________    Canola    6%        58%         36%    Soybean  15%        24%         61%    Corn     13%        25%         62%    Peanut   18%        48%         34%    Safflower              9%        13%         78%    Sunflower              9%        41%         51%    Cotton   30%        19%         51%    ______________________________________

Many recent research efforts have examined the role that saturated andunsaturated fatty acids play in reducing the risk of coronary heartdisease. In the past, it was believed that mono-unsaturates, in contrastto saturates and poly-unsaturates, had no effect on serum cholesteroland coronary heart disease risk. Several recent human clinical studiessuggest that diets high in mono-unsaturated fat and low in saturated fatmay reduce the "bad" (low-density lipoprotein) cholesterol whilemaintaining the "good" (high-density lipoprotein) cholesterol (Mattsonet al., Journal of Lipid Research (1985) 26:194-202). Soybean oil ishigh in saturated fatty acids when compared to other sources ofvegetable oil and contains a low proportion of oleic acid, relative tothe total fatty acid content of the soybean seed. These characteristicsdo not meet important health needs as defined by the American HeartAssociation.

A soybean oil low in total saturates and polyunsaturates and high inmonounsaturate would provide significant health benefits to the UnitedStates population, as well as, economic benefit to oil processors.

Oil biosynthesis in plants has been fairly well-studied see Harwood(1989) in Critical Reviews in Plant Sciences, Vol. 8 (1):1-43!. Thebiosynthesis of palmitic, stearic and oleic acids occur in the plastidsby the interplay of three key enzymes of the "ACP track": palmitoyl-ACPelongase, stearoyl-ACP desaturase and the acyl-ACP thioesterases.

Of these three enzyme types, the acyl-ACP thioesterases function toremove the acyl chain from the carrier protein (ACP) and thus from themetabolic pathway. The oleoy-ACP thioesterase catalyzes the hydrolysisof oleoyl-ACP thioesters at high rates and at much lower rates thehydrolysis of palmitoyl-ACP and stearoyl-ACP. This multiple activityleads to substrate competition between enzymes and it is the competitionof this acyl-ACP thioesterase and palmitoyl-ACP elongase for the samesubstrate and of acyl-ACP thioesterase and stearoyl-ACP desaturase forthe same substrate that leads to a portion of the production of thepalmitic and stearic acids found in the triacylglyceride of vegetableoils.

Once removed from the ACP track fatty acids are exported to thecytoplasm and there used to synthesize acyl-coenzyme A. These acyl-CoA'sare the acyl donors for at least three different glycerol acylatingenzymes (glycerol-3-P acyltransferase, 1-acyl-glycerol-3-Pacyltransferase and diacylglycerol acyltransferase) which incorporatethe acyl moieties into triacylglycerides during oil biosynthesis.

These acyltransferases show a strong, but not absolute, preference forincorporating saturated fatty acids at positions 1 and 3 andmonounsaturated fatty acid at position 2 of the triglyceride. Thus,altering the fatty acid composition of the acyl pool will drive by massaction a corresponding change in the fatty acid composition of the oil.

Based on the above discussion, one approach to altering the levels ofpalmitic, stearic and oleic acids in vegetable oils is by altering theirlevels in the cytoplasmic acyl-CoA pool used for oil biosynthesis.

In previous work (WO 9211373) Applicant has demonstrated that oleoyl-ACPthioesterase may be modulated using cloned cDNA encoding the soybeanenzyme. Oleoyl-ACP thioesterase cDNA was used to form chimeric genes forthe transformation of soybean plant cells resulting in the anti-senseinhibition of acyl-ACP thioesterase in the plant seed.

Applicant has now discovered an entirely new plant thioesterase withactivity on a C16 substrate that is also useful for the regulation ofthe acyl coenzyme A pool. Applicant has isolated nucleic acid fragmentsthat encode soybean and canola palmitoyl-ACP thioesterases that areuseful in modifying fatty acid composition in oil-producing species bygenetic transformation. Thus, transfer of the nucleic acid fragments ofthe invention or a part thereof that encodes a functional enzyme, alongwith suitable regulatory sequences that direct the transcription oftheir mRNA, into a living cell will result in the production orover-production of palmitoyl-ACP thioesterases and will result inincreased levels of saturated fatty acids in cellular lipids, includingtriacylglycerols.

Transfer of the nucleic acid fragments of the invention or a partthereof, along with suitable regulatory sequences that direct thetranscription of their anti-sense RNA, into plants will result in theinhibition of expression of the endogenous palmitoyl-ACP thioesterasethat is substantially homologous with the transferred nucleic acidfragment and will result in decreased levels of saturated fatty acids incellular lipids, including triacylglycerols.

Transfer of the nucleic acid fragments of the invention or a partthereof, along with suitable regulatory sequences that direct thetranscription of their mRNA, into plants may result in inhibition bycosuppression of the expression of the endogenous palmitoyl-ACPthioesterase gene that is substantially homologous with the transferrednucleic acid fragment and may result in decreased levels of unsaturatedfatty acids in cellular lipids, including triacylglycerols.

SUMMARY OF THE INVENTION

A means to control the levels of saturated and unsaturated fatty acidsin edible plant oils has been discovered. Utilizing the soybean seedpalmitoyl-ACP thioesterase cDNA, for either the precursor or enzyme,chimeric genes are created and may be utilized to transform soybeanplants to produce seed oils with reduced levels of saturated fattyacids. Similarly the canola seed palmitoyl-ACP thioesterase cDNA foreither the precursor or enzyme may be utilized to create chimeric genesand these genes may then be used to transform canola plants to produceseed oils with reduced levels of saturated fatty acids.

Specifically, one aspect of the present invention is a nucleic acidfragment comprising a nucleotide sequence encoding the soybean seedpalmitoyl-ACP thioesterase cDNA corresponding to nucleotides 1 to 1688in the sequence shown in Sequence Description SEQ ID NO:1, or anynucleic acid fragment substantially homologous therewith. In addition,another aspect involves a nucleic acid fragment comprising a nucleotidesequence encoding the canola seed palmitoyl-ACP thioesterase cDNAcorresponding to the nucleotides 1 to 1488 in the Sequence DescriptionSEQ ID NO:2, nucleotides 1 to 1674 in the Sequence Description SEQ IDNO:31 or any nucleic acid fragment substantially homologous therewith.Preferred are those nucleic acid fragments encoding the soybean seedpalmitoyl-ACP thioesterase precursor, the mature soybean seedpalmitoyl-ACP thioesterase enzyme, the canola seed palmitoyl-ACPthioesterase precursor, and the mature canola seed palmitoyl-ACPthioesterase enzyme.

Another aspect of this invention involves a chimeric gene capable oftransforming a soybean plant cell comprising a nucleic acid fragmentencoding the soybean seed palmitoyl-ACP thioesterase cDNA of Sequence ID1 operably linked to suitable regulatory sequences producing anti-senseinhibition of soybean seed palmitoyl-ACP thioesterase in the seed orlinked suitably to produce sense expression of the soybean seedpalmitoyl-ACP thioesterase gene resulting in either over expression ofthe palmitoyl-ACP thioesterase protein or under expression of thepalmitoyl-ACP thioesterase protein when co-suppression occurs. Preferredare those chimeric genes which incorporate nucleic acid fragmentsencoding soybean seed palmitoyl-ACP thioesterase precursor or maturesoybean seed palmitoyl-ACP thioesterase enzyme.

Yet another embodiment of the invention involves a method of producingseed oil containing either elevated or reduced levels of saturated fattyacids comprising: (a) transforming a soybean plant cell with a chimericgene described above, (b) growing sexually mature plants from saidtransformed plant cells, (c) screening progeny seeds from said sexuallymature plants for the desired levels of palmitic and stearic acid, and(d) crushing said progeny seed to obtain said oil containing decreasedlevels of palmitic and stearic acid. Preferred methods of transformingsuch plant cells would include the use of Ti and Ri plasmids ofAgrobacterium, electroporation, and high-velocity ballistic bombardment.

Another aspect of this invention involves a chimeric gene capable oftransforming a canola plant cell comprising a nucleic acid fragmentencoding the canola seed palmitoyl-ACP thioesterase cDNA of Sequence ID2 or Sequence ID 31 operably linked to suitable regulatory sequencesproducing anti-sense inhibition of canola seed palmitoyl-ACPthioesterase in the seed or linked suitably to produce sense expressionof the canola seed palmitoyl-ACP thioesterase gene resulting in eitherover expression of the palmitoyl-ACP thioesterase protein or underexpression of the palmitoyl-ACP thioesterase protein when co-suppressionoccurs. Preferred are those chimeric genes which incorporate nucleicacid fragments encoding canola seed palmitoyl-ACP thioesterase precursoror mature canola seed palmitoyl-ACP thioesterase enzyme.

BRIEF DESCRIPTION OF THE SEQUENCES

The invention can be more fully understood from the following detaileddescription and the Sequence Descriptions which form a part of thisapplication.

The sequence descriptions summarize the Sequences Listing attachedhereto. The Sequence Listing contains one letter codes for nucleotidesequence characters and the three letter codes for amino acids asdefined in the IUPAC-IUB standards described in Nucleic Acids Research13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373(1984), and the symbols and format used for all nucleotide and aminoacid sequence data further comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

SEQ ID NO:1 sets forth the nucleotide sequence of a soybeanpalmitoyl-ACP thioesterase cDNA.

SEQ ID NO:2 sets forth the nucleotide sequence of a canola palmitoyl-ACPthioesterase cDNA.

SEQ ID NOS:3 and 4 set forth the sequence of oligonucleotides used toform a linker.

SEQ ID NO:5 sets forth the sequence of an oligonucleotide primer derivedfrom SEQ ID NO: 1.

SEQ ID NO:6 sets forth the deduced sequence of the protein expressed inE. coli from the canola cDNA set forth in SEQ ID NO:2.

SEQ ID NO:7 is the deduced sequence of the protein expressed in E. colifrom the soybean cDNA set forth in SEQ ID NO:1.

SEQ ID NO:8 sets forth the nucleotide sequence of the napin promoterused to drive seed-specific expression of thioesterase in canola.

SEQ ID NO:9 sets forth the complement of the sequence of SEQ ID NO:8.

SEQ ID NO:10 sets forth the sequence derived from the napin gene 3' ofthe coding region.

SEQ ID NO:11 sets forth the complement of the sequence of SEQ ID NO:10.

SEQ ID NO:12 sets forth the sequence of an oligonucleotide primerderived from SEQ ID NO:1.

SEQ ID NOS:13 through 20 set forth the sequences of oligonucleotideprimers used to amplify segments of the napin promoter.

SEQ ID NOS:21 and 22 set forth the sequences of oligonucleotide primersused to amplify segments of the napin terminator region.

SEQ ID NOS:23 and 24 set forth the sequences of oligonucleotide primersused to amplify a segment from the 3' end of the napin promoter.

SEQ ID NOS:25 and 26 set forth short sequences of DNA that wereintroduced into the PCR amplified version of the napin terminator.

SEQ ID NO:27 sets forth the reverse complement of the soybean cDNAsequence of SEQ ID NO:1.

SEQ ID NO:28 sets forth the reverse complement of the canola cDNAsequence of SEQ ID NO:2.

SEQ ID NO:29 sets forth the predicted amino acid sequence encoded by thesequence of SEQ ID NO:1.

SEQ ID NO:30 sets forth the predicted amino acid sequence encoded by thesequence of SEQ ID NO:2.

SEQ ID NO:31 sets forth the nucleotide sequence of a second canolapalmitoyl-ACP thioesterase cDNA.

SEQ ID NO:32 sets forth the predicted amino acid sequence encoded by thesequence of SEQ ID NO:31.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be used.

Fatty acids are specified by the number of carbon atoms and the numberand position of the double bond: the numbers before and after the colonrefer to the chain length and the number of double bonds, respectively.The number following the fatty acid designation indicates the positionof the double bond from the carboxyl end of the fatty acid with the "c"affix for the cis-configuration of the double bond. For example,palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1,9c),petroselinic acid (18:1, 6c), linoleic acid (18:2,9c,12c), g-linolenicacid (18:3, 6c,9c,12c) and a-linolenic acid (18:3, 9c,12c,15c). Unlessotherwise specified 18:1, 18:2 and 18:3 refer to oleic, linoleic andlinolenic fatty acids. The term "palmitoyl-ACP thioesterase" used hereinrefers to an enzyme which catalyzes the hydrolytic cleavage of thecarbon-sulfur thioester bond in the pantothene prosthetic group ofpalmitoyl-acyl carrier protein as its preferred reaction. Hydrolysis ofother fatty acid-acyl carrier protein thioesters may also be catalyzedby the enzymes. The term "nucleic acid" refers to a large molecule whichcan be single-stranded or double-stranded, composed of monomers(nucleotides) containing a sugar, a phosphate and either a purine orpyrimidine. A "nucleic acid fragment" is a fraction of a given nucleicacid molecule. In higher plants, deoxyribonucleic acid (DNA) is thegenetic material while ribonucleic acid (RNA) is involved in thetransfer of the information in DNA into proteins. A "genome" is theentire body of genetic material contained in each cell of an organism.The term "nucleotide sequence" refers to the sequence of DNA or RNApolymers, which can be single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The term "oligomer" refers toshort nucleotide sequences, usually up to 100 bases long. As usedherein, the term "homologous to" refers to the relatedness between thenucleotide sequence of two nucleic acid molecules or between the aminoacid sequences of two protein molecules. Estimates of such homology areprovided by either DNA-DNA or DNA-RNA hybridization under conditions ofstringency as is well understood by those skilled in the art (Hames andHiggins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.); or by the comparison of sequence similarity between two nucleicacids or proteins, such as by the method of Needleman et al. (J. Mol.Biol. (1970) 48:443-453). As used herein, "substantially homologous"refers to nucleotide sequences that have more than 90% overall identityat the nucleotide level with the coding region of the claimed sequence,such as genes and pseudo-genes corresponding to the coding regions. Thenucleic acid fragments described herein include molecules which comprisepossible variations, both man-made and natural, such as but not limitedto (a) those that involve base changes that do not cause a change in anencoded amino acid, or (b) which involve base changes that alter anamino acid but do not affect the functional properties of the proteinencoded by the DNA sequence, (c) those derived from deletions,rearrangements, amplifications, random or controlled mutagenesis of thenucleic acid fragment, and (d) even occasional nucleotide sequencingerrors.

"Gene" refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5' non-coding) andfollowing (3' non-coding) the coding region. "Native" gene refers to anisolated gene with its own regulatory sequences as found in nature."Chimeric gene" refers to a gene that comprises heterogeneous regulatoryand coding sequences not found in nature. "Endogenous" gene refers tothe native gene normally found in its natural location in the genome andis not isolated. A "foreign" gene refers to a gene not normally found inthe host organism but that is introduced by gene transfer. "Pseudo-gene"refers to a genomic nucleotide sequence that does not encode afunctional enzyme.

"Coding sequence" refers to a DNA sequence that codes for a specificprotein and excludes the non-coding sequences. It may constitute an"uninterrupted coding sequence", i.e., lacking an intron or it mayinclude one or more introns bounded by appropriate splice junctions. An"intron" is a nucleotide sequence that is transcribed in the primarytranscript but that is removed through cleavage and re-ligation of theRNA within the cell to create the mature mRNA that can be translatedinto a protein.

"Initiation codon" and "termination codon" refer to a unit of threeadjacent nucleotides in a coding sequence that specifies initiation andchain termination, respectively, of protein synthesis (mRNAtranslation). "Open reading frame" refers to the coding sequenceuninterrupted by introns between initiation and termination codons thatencodes an amino acid sequence.

"RNA transcript" refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. "Messenger RNA (mRNA)" refers tothe RNA that is without introns and that can be translated into proteinby the cell. "cDNA" refers to a double-stranded DNA that iscomplementary to and derived from mRNA. "Sense" RNA refers to RNAtranscript that includes the mRNA. "Antisense RNA" refers to a RNAtranscript that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene byinterfering with the processing, transport and/or translation of itsprimary transcript or mRNA. The complementarity of an antisense RNA maybe with any part of the specific gene transcript, i.e., at the 5'non-coding sequence, 3' non-coding sequence, introns, or the codingsequence. In addition, as used herein, antisense RNA may contain regionsof ribozyme sequences that increase the efficacy of antisense RNA toblock gene expression. "Ribozyme" refers to a catalytic RNA and includessequence-specific endoribonucleases.

As used herein, "suitable regulatory sequences" refer to nucleotidesequences in native or chimeric genes that are located upstream (5'),within, and/or downstream (3') to the nucleic acid fragments of theinvention, which control the expression of the nucleic acid fragments ofthe invention. The term "expression", as used herein, refers to thetranscription and stable accumulation of the sense (mRNA) or theantisense RNA derived from the nucleic acid fragment(s) of the inventionthat, in conjunction with the protein apparatus of the cell, results inaltered levels of the palmitoyl-ACP thioesterase. Expression oroverexpression of the gene involves transcription of the gene andtranslation of the mRNA into precursor or mature palmitoyl-ACPthioesteras proteins. "Antisense inhibition" refers to the production ofantisense RNA transcripts capable of preventing the expression of thetarget protein. "Overexpression" refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms. "Cosuppression" refers to theexpression of a foreign gene which has substantial homology to anendogenous gene resulting in the suppression of expression of both theforeign and the endogenous gene. "Altered levels" refers to theproduction of gene product(s) in transgenic organisms in amounts orproportions that differ from that of normal or non-transformedorganisms.

"Promoter" refers to a DNA sequence in a gene, usually upstream (5') toits coding sequence, which controls the expression of the codingsequence by providing the recognition for RNA polymerase and otherfactors required for proper transcription. In artificial DNA constructspromoters can also be used to transcribe antisense RNA. Promoters mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions. It may alsocontain enhancer elements. An "enhancer" is a DNA sequence which canstimulate promoter activity. It may be an innate element of the promoteror a heterologous element inserted to enhance the level and/ortissue-specificity of a promoter. "Constitutive promoters" refers tothose that direct gene expression in all tissues and at all times."Tissue-specific" or "development-specific" promoters as referred toherein are those that direct gene expression almost exclusively inspecific tissues, such as leaves or seeds, or at specific developmentstages in a tissue, such as in early or late embryogenesis,respectively.

The "3' non-coding sequences" refers to the DNA sequence portion of agene that contains a poly-adenylation signal and any other regulatorysignal capable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3' end of the mRNAprecursor.

"Transformation" herein refers to the transfer of a foreign gene intothe genome of a host organism and its genetically stable inheritance."Restriction fragment length polymorphism" refers to different sizedrestriction fragment lengths due to altered nucleotide sequences in oraround variant forms of genes. "Fertile" refers to plants that are ableto propagate sexually.

"Plants" refer to photosynthetic organisms, both eukaryotic andprokaryotic, whereas the term "Higher plants" refers to eukaryoticplants. "Oil-producing species" herein refers to plant species whichproduce and store triacylglycerol in specific organs, primarily inseeds. Such species include soybean (Glycine max), rapeseed and canola(including Brassica napus, B. campestris), sunflower (Helianthus annus),cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao),safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconutpalm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinuscommunis) and peanut (Arachis hypogaea). The group also includesnon-agronomic species which are useful in developing appropriateexpression vectors such as tobacco, rapid cycling Brassica species, andArabidopsis thaliana, and wild species which may be a source of uniquefatty acids.

"Sequence-dependent protocols" refer to techniques that rely on anucleotide sequence for their utility. Examples of sequence-dependentprotocols include, but are not limited to, the methods of nucleic acidand oligomer hybridization and methods of DNA and RNA amplification suchas are exemplified in various uses of the polymerase chain reaction(PCR).

"PCR" or "polymerase chain reaction" will refer to a method that resultsin the linear or logarithmic amplification of nucleic acid molecules.PCR generally requires a replication composition consisting of, forexample, nucleotide triphosphates, two primers with appropriatesequences, DNA or RNA polymerase and proteins. These reagents anddetails describing procedures for their use in amplifying nucleic acidsare provided in U.S. Pat. No. 4,683,202 (1987, Mullis, et al.) and U.S.Pat. No. 4,683,195 (1986, Mullis, et al.).

The present invention describes two nucleic acid fragments that encodesoybean and canola seed palmitoyl-ACP thioesterases. These enzymescatalyze the hydrolytic cleavings of palmitic acid, stearic acid andoleic acid from ACP in the respective acyl-ACPs. Transfer of one or bothof these nucleic acid fragments of the invention or a part thereof thatencodes a functional enzyme, with suitable regulatory sequences into aliving cell will result in the production or over-production ofpalmitoly-ACP thioesterase, which may result in increased levels ofpalmitic and to a lesser extent, stearic acids in cellular lipids,including oil.

Transfer of the nucleic acid fragment or fragments of the invention,with suitable regulatory sequences that transcribe the present cDNA,into a plant which has an endogenous seed palmitoyl-ACP thioesterasethat is substantially homogeneous with the present cDNA may result ininhibition by co-suppresion of the expression of the endogenouspalmitoyl-ACP thioesterase gene and, consequently, in a decreased amountof palmitic and to a lesser extent stearic acids in the seed oil.

Transfer of the nucleic acid fragment or fragments of the invention intoa soybean or canola plants with suitable regulatory sequences thattranscribe the anti-sense RNA complementary to the mRNA, or itsprecursor, for seed palmitoyl-ACP thioesterase may result in theinhibition of the expression of the endogenous palmitoyl-ACPthioesterase gene and, consequently, in reduced amounts of palmitic andto a lesser extent stearic acids in the seed oil.

The nucleic acid fragments of the invention can also be used as arestriction fragment length polymorphism markers in soybean and canolagenetic studies and breeding programs.

Identification and Isolation of Soybean and Canola Palmitoyl-ACPThioesterase Coding cDNA

In order to identify cDNA encoding for palmitoyl-ACP thioesterase inboth soybean and canola it was first necessary to construct a probesuitable for screening cDNA libraries from these plant genomes. Aportion of the Arabidopis cDNA known to have significant homology withan Umbellularia C12:0-ACP thioesterase was used to design PCR primers(SEQ ID NO:3 and 4). Polysomal RNA was isolated and purified fromArabidopis and used as a template for RNA-PCR (GeneAmp® PNA-PCR kitPerkin Elmer Cetus, part number N808-0017). Using this method a 560 bpfragment was generated, and radiolabeled to be used as a probe forscreening soybean and canola cDNA libraries.

Methods of creating cDNA libraries from eukaryotic genomes are wellknown in the art (see, for example, Sambrook, et al. (Molecular Cloning,A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor LaboratoryPress). In a preferred method total RNA is isolated (Kamalay et al.,(Cell (1980) 19:935-946) and polyadenylated mRNA is purified by standardmeans. mRNA is incorporated into a suitable phage such as lambda phageand used to transform a suitable host such as E. coli. Transformedclones are screened for positively hybridizing plaques using theradio-labelled, PCR derived probe.

In this manner DNA fragments were selected from both soybean and canolathat had potential for encoding an acyl-ACP thioesterase. The DNAfragment isolated from soybean is identified as SEQ ID NO:1 and the DNAfragments isolated from canola are identified as SEQ ID NO:2 and SEQ IDNO:31.

Expression of Soybean and Canola Acyl-ACP Thioesterase Encoding DNA inE. coli

In order to verify the function of the isolated soybean and canola DNAfragments it was necessary to express the fragments in recombinant hostsfor protein purification and analysis of enzyme activity.

The present invention provides vectors and host cells suitable forgenetic manipulations and the expression of recombinant proteins.Suitable hosts may include a variety of gram negative and gram positivebacteria where E. coli is generally preferred. Examples ofbacteria-derived vectors include plasmid vectors such as pBR322, pUC19,pSP64, pUR278 and pORF1. Illustrative of suitable viral vectors arethose derived from phage, vaccinia, and a variety of viruses. Examplesof phage vectors include 1⁺, 1EMBL3, 12001, 1gt10, 1gt11, Charon 4a,Charon 40, and 1ZAP/R. pXB3 and pSC11 are exemplary of vaccinia vectors(Chakrabarti et al., Molec. Cell. Biol. 5:3401-9 (1985) and Mackett etal. J. Virol. 49:857864 (1984). Preferred in the present invention arethe bacteria derived vectors such as pET-3d (described by F. W. Studier,A. H. Rosenberg, J. J. Dunn and J. W. Dubendorff, Methods in EnzymologyVol. 185) and the host E. coli strain BL21(DE3)(pLysE).

Once suitable vectors are constructed they are used to transformsuitable bacterial hosts. Introduction of desired DNA fragments into E.coli may be accomplished by known procedures such as by transformation,e.g., using calcium-permeabilized cells, electroporation, or bytransfection using a recombinant phage virus. (Sambrook et al., supra.)

For the expression of the soybean and canola DNA fragments (SEQ ID NO:1and 2, respectively) the fragments were first cut with the appropriaterestriction enzymes for the isolation of the region encoding the matureprotein. Following this the restriction fragments were ligated to anappropriate linker sequence and inserted into a suitable vectordownstream of an appropriate promoter. Suitable promoters may be eitherinducible or constitutive and are preferably derived from bacteria.Examples of suitable promoters are T7 and lac.

Thioesterase Assay:

Methods for the measurement of thioesterase activity are known in theart (see, for example, Smith et al., Biochem, J. 212, 155, (1983) andSpencer et al., J. Biol. Chem., 253, 5922, (1978)). For the purpose ofthe present invention a modification of the method of Mckeon and StumpfJ. Biol. Chem. (1982) 257:12141-12147! was used involving the synthesisof radiolabelled substrate ( ¹⁴ C!acyl-ACP) using ACP and ACP synthetaseisolated from E. coli. Solutions of ¹⁴ C! palmitic acid, ¹⁴ C! stearicacid, ¹⁴ C! oleic acid, ¹⁴ C! lauric acid, and ¹⁴ C! decanoic acid wereadded to purified ACP in the presence of ACP synthetase and theresulting radiolabelled acyl ACP was purified by standard methods.Activity of the protein encoded and expressed by SEQ ID NO:1 and SEQ IDNO:2 was measured on the basis of the amount of ¹⁴ C! substrate that washydrolyzed.

Inhibition of Plant Target Genes by Use of Antisense RNA

Antisense RNA has been used to inhibit plant target genes in atissue-specific manner (see van der Krol et al., Biotechniques (1988)6:958-976). Antisense inhibition has been shown using the entire cDNAsequence (Sheehy et al., Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809)as well as a partial cDNA sequence (Cannon et al., Plant Molec. Biol.(1990) 15:39-47). There is also evidence that the 3' non-codingsequences (Ch'ng et al., Proc. Natl. Acad. Sci. USA (1989)86:10006-10010) and fragments of 5' coding sequence, containing as fewas 41 base-pairs of a 1.87 kb cDNA (Cannon et al., Plant Molec. Biol.(1990) 15:39-47), can play important roles in anti-sense inhibition.

The entire soybean palmitoyl-ACP thioesterase cDNA was cloned in theanti-sense orientation with respect to a soybean β-conglycinin promoterand the chimeric gene transformed into soybean somatic embryos. Asdemonstrated in Example 2, these embryos serve as good model system forsoybean zygotic embryos. Transformed somatic embryos showed inhibitionof palmitate and possibly stearate biosyntheis. Similarly, the entireBrassica napus palmitoyl-ACP cDNA was cloned in the anti-senseorientation with respect to a rapeseed napin promoter and the chimericgene transformed into B. napus.

Inhibition of Plant Target Genes by Cosuppression

The phenomenon of cosuppression has also been used to inhibit planttarget genes in a tissue-specific manner. Cosuppression of an endogenousgene using the entire cDNA sequence (Napoli et al., The Plant Cell(1990) 2:279-289; van der Krol et al., The Plant Cell (1990) 2:291-299)as well as a partial cDNA sequence (730 bp of a 1770 bp cDNA) (Smith etal., Mol. Gen. Genetics (1990) 224:477-481) are known.

The nucleic acid fragments of the instant invention encodingpalmitoyl-ACP thioesterases or parts thereof, with suitable regulatorysequences, can be used to reduce the level of palmitoyl-ACPthioesterase, thereby altering fatty acid composition, in transgenicplants which contain an endogenous gene substantially homologous to theintroduced nucleic acid fragment. The experimental procedures necessaryfor this are similar to those described above for the anti-senseexpression of palmitoyl-ACP thioesterase nucleic acid fragments exceptthat one may use a either whole or partial cDNA.

Endogenous genes can also be inhibited by non-coding regions of anintroduced copy of the gene for example, Brusslan, J. A., et al. (1993)Plant Cell 5:667-677; Matzke, M. A. et al Plant Molecular Biology16:821-830!.

Selection of Hosts, Promoters and Enhancers

A preferred class of heterologous hosts for the expression of thenucleic acid fragments of the invention are eukaryotic hosts,particularly the cells of higher plants. Particularly preferred amongthe higher plants are the oil-producing species, such as soybean(Glycine max), rapeseed (including Brassica napus, B. campestris),sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zeamays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oilpalm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linumusitatissimum), and peanut (Arachis hypogaea).

Expression in plants will use regulatory sequences functional in suchplants. The expression of foreign genes in plants is well-established(De Blaere et al., Meth. Enzymol. (1987) 153:277-291). The source of thepromoter chosen to drive the expression of the fragments of theinvention is not critical provided it has sufficient transcriptionalactivity to accomplish the invention by increasing or decreasing,respectively, the level of translatable mRNA for the fatty aciddesaturases in the desired host tissue. Preferred promoters include (a)strong constitutive plant promoters, such as those directing the 19S and35S transcripts in cauliflower mosaic virus (Odell et al., Nature (1985)313:810-812; Hull et al., Virology (1987) 86:482-493), (b) tissue- ordevelopmentally-specific promoters, and (c) other transcriptionalpromoter systems engineered in plants, such as those using bacteriophageT7 RNA polymerase promoter sequences to express foreign genes. Examplesof tissue-specific promoters are the light-inducible promoter of thesmall subunit of ribulose 1,5-bis-phosphate carboxylase (if expressionis desired in photosynthetic tissues), the maize zein protein promoter(Matzke et al., EMBO J. (1984) 3:1525-1532), and the chlorophyll a/bbinding protein promoter (Lampa et al., Nature (1986) 316:750-752).

Particularly preferred promoters are those that allow seed-specificexpression. This may be especially useful since seeds are the primarysource of vegetable oils and also since seed-specific expression willavoid any potential deleterious effect in non-seed tissues. Examples ofseed-specific promoters include, but are not limited to, the promotersof seed storage proteins, which can represent up to 90% of total seedprotein in many plants. The seed storage proteins are strictlyregulated, being expressed almost exclusively in seeds in a highlytissue-specific and stage-specific manner (Higgins et al., Ann. Rev.Plant Physiol. (1984) 35:191-221; Goldberg et al., Cell (1989)56:149-160). Moreover, different seed storage proteins may be expressedat different stages of seed development.

Expression of seed-specific genes has been studied in great detail (seereviews by Goldberg et al., Cell (1989) 56:149-160 and Higgins et al.,Ann. Rev. Plant Physiol. (1984) 35:191-221). There are currentlynumerous examples of seed-specific expression of seed storage proteingenes in transgenic dicotyledonous plants. These include genes fromdicotyledonous plants for bean b-phaseolin (Sengupta-Gopalan et al.,Proc. Natl. Acad. Sci. USA (1985) 82:3320-3324; Hoffman et al., PlantMol. Biol. (1988) 11:717-729), bean lectin (Voelker et al., EMBO J.(1987) 6:3571-3577), soybean lectin (Okamuro et al., Proc. Natl. Acad.Sci. USA (1986) 83:8240-8244), soybean Kunitz trypsin inhibitor(Perez-Grau et al., Plant Cell (1989) 1:095-1109), soybean b-conglycinin(Beachy et al., EMBO J. (1985) 4:3047-3053; pea vicilin (Higgins et al.,Plant Mol. Biol. (1988) 11:683-695), pea convicilin (Newbigin et al.,Planta (1990) 180:461-470), pea legumin (Shirsat et al., Mol. Gen.Genetics (1989) 215:326-331); rapeseed napin (Radke et al., Theor. Appl.Genet. (1988) 75:685-694) as well as genes from monocotyledonous plantssuch as for maize 15 kD zein (Hoffman et al., EMBO J. (1987)6:3213-3221), maize 18 kD oleosin (Lee et al., Proc. Natl. Acad. Sci.USA (1991) 888:6181-6185), barley b-hordein (Marris et al., Plant Mol.Biol. (1988) 10:359-366) and wheat glutenin (Colot et al., EMBO J.(1987) 6:3559-3564). Moreover, promoters of seed-specific genes operablylinked to heterologous coding sequences in chimeric gene constructs alsomaintain their temporal and spatial expression pattern in transgenicplants. Such examples include use of Arabidopsis thaliana 2S seedstorage protein gene promoter to express enkephalin peptides inArabidopsis and B. napus seeds (Vandekerckhove et al., Bio/Technology(1989) 7:929-932), bean lectin and bean b-phaseolin promoters to expressluciferase (Riggs et al., Plant Sci. (1989) 63:47-57), and wheatglutenin promoters to express chloramphenicol acetyl transferase (Colotet al., EMBO J. (1987) 6:3559-3564).

Of particular use in the expression of the nucleic acid fragment of theinvention will be the heterologous promoters from several soybean seedstorage protein genes such as those for the Kunitz trypsin inhibitor(Jofuku et al., Plant Cell (1989) 1:1079-1093; glycinin (Nielson et al.,Plant Cell (1989) 1:313-328), and b-conglycinin (Harada et al., PlantCell (1989) 1:415-425). Promoters of genes for a- and b-subunits ofsoybean b-conglycinin storage protein will be particularly useful inexpressing the mRNA or the antisense RNA in the cotyledons at mid- tolate-stages of seed development (Beachy et al., EMBO J. (1985)4:3047-3053) in transgenic plants. This is because there is very littleposition effect on their expression in transgenic seeds, and the twopromoters show different temporal regulation. The promoter for thea-subunit gene is expressed a few days before that for the b-subunitgene. This is important for transforming rapeseed where oil biosynthesisbegins about a week before seed storage protein synthesis (Murphy etal., J. Plant Physiol. (1989) 135:63-69).

Also of particular use will be promoters of genes expressed during earlyembryogenesis and oil biosynthesis. The native regulatory sequences,including the native promoters, of the palmitoyl-ACP thioesterase genesexpressing the nucleic acid fragments of the invention can be usedfollowing their isolation by those skilled in the art. Heterologouspromoters from other genes involved in seed oil biosynthesis, such asthose for B. napus isocitrate lyase and malate synthase (Comai et al.,Plant Cell (1989) 1:293-300), delta-9 desaturase from safflower(Thompson et al. Proc. Natl. Acad. Sci. USA (1991) 88:2578-2582) andcastor (Shanklin et al., Proc. Natl. Acad. Sci. USA (1991)88:2510-2514), acyl carrier protein (ACP) from Arabidopsis(Post-Beittenmiller et al., Nucl. Acids Res. (1989) 17:1777), B. napus(Safford et al., Eur. J. Biochem. (1988) 174:287-295), and B. campestris(Rose et al., Nucl. Acids Res. (1987) 15:7197), b-ketoacyl-ACPsynthetase from barley (Siggaard-Andersen et al., Proc. Natl. Acad. Sci.USA (1991) 88:4114-4118), and oleosin from Zea mays (Lee et al., Proc.Natl. Acad. Sci. USA (1991) 88:6181-6185), soybean (Genbank AccessionNo: X60773) and B. napus (Lee et al., Plant Physiol. (1991)96:1395-1397) will be of use. If the sequence of the corresponding genesis not disclosed or their promoter region is not identified, one skilledin the art can use the published sequence to isolate the correspondinggene and a fragment thereof containing the promoter. The partial proteinsequences for the relatively-abundant enoyl-ACP reductase and acetyl-CoAcarboxylase are also published (Slabas et al., Biochim. Biophys. Acta(1987) 877:271-280; Cottingham et al., Biochim. Biophys. Acta (1988)954:201-207) and one skilled in the art can use these sequences toisolate the corresponding seed genes with their promoters. Attaining theproper level of expression of the nucleic acid fragments of theinvention may require the use of different chimeric genes utilizingdifferent promoters. Such chimeric genes can be transferred into hostplants either together in a single expression vector or sequentiallyusing more than one vector.

It is envisioned that the introduction of enhancers or enhancer-likeelements into the promoter regions of either the native or chimericnucleic acid fragments of the invention will result in increasedexpression to accomplish the invention. This would include viralenhancers such as that found in the 35S promoter (Odell et al., PlantMol. Biol. (1988) 10:263-272), enhancers from the opine genes (Fromm etal., Plant Cell (1989) 1:977-984), or enhancers from any other sourcethat result in increased transcription when placed into a promoteroperably linked to the nucleic acid fragment of the invention.

Of particular importance is the DNA sequence element isolated from thegene for the a-subunit of b-conglycinin that can confer 40-foldseed-specific enhancement to a constitutive promoter (Chen et al., Dev.Genet. (1989) 10:112-122). One skilled in the art can readily isolatethis element and insert it within the promoter region of any gene inorder to obtain seed-specific enhanced expression with the promoter intransgenic plants. Insertion of such an element in any seed-specificgene that is expressed at different times than the b-conglycinin genewill result in expression in transgenic plants for a longer periodduring seed development.

Any 3' non-coding region capable of providing a polyadenylation signaland other regulatory sequences that may be required for the properexpression of the nucleic acid fragments of the invention can be used toaccomplish the invention. This would include 3' ends of the native fattyacid desaturase(s), viral genes such as from the 35S or the 19Scauliflower mosaic virus transcripts, from the opine synthesis genes,ribulose 1,5-bisphosphate carboxylase, or chlorophyll a/b bindingprotein. There are numerous examples in the art that teach theusefulness of different 3' non-coding regions.

Transformation Methods

Various methods of transforming cells of higher plants according to thepresent invention are available to those skilled in the art (see EPOPub. 0 295 959 A2 and 0 318 341 A1). Such methods include those based ontransformation vectors utilizing the Ti and Ri plasmids of Agrobacteriumspp. It is particularly preferred to use the binary type of thesevectors. Ti-derived vectors transform a wide variety of higher plants,including monocotyledonous and dicotyledonous plants (Sukhapinda et al.,Plant Mol. Biol. (1987) 8:209-216; Potrykus, Mol. Gen. Genet. (1985)199:183). Other transformation methods are available to those skilled inthe art, such as direct uptake of foreign DNA constructs (see EPO Pub. 0295 959 A2), techniques of electroporation (Fromm et al., Nature (1986)(London) 319:791) or high-velocity ballistic bombardment with metalparticles coated with the nucleic acid constructs (Kline et al., Nature(1987) (London) 327:70). Once transformed, the cells can be regeneratedby those skilled in the art.

Of particular relevance are the recently described methods to transformforeign genes into commercially important crops, such as rapeseed (DeBlock et al., Plant Physiol. (1989) 91:694-701), sunflower (Everett etal., Bio/Technology (1987) 5:1201), and soybean (Christou et al., Proc.Natl. Acad. Sci USA (1989) 86:7500-7504.

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

EXAMPLES MATERIALS AND METHODS

Various solutions used in the experimental manipulations are referred toby their common names such as "SSC", "SSPE", "Denhardt's solution", etc.The composition of these solutions as well as any method for thestandard manipulation of nucleic acids, transformatins and growth of E.coli may be found by reference to Sambrook, et al. (Molecular Cloning, ALaboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press)

Growth Media:

Media for the growth of plant embryo cultures is given below:

    ______________________________________    Plant Embryo Culture Media    ______________________________________    Media:    SB55 and SBP6 Stock Solutions (g/L):    MS Sulfate 100X Stock    MgSO.sub.4 7H.sub.2 O 37.0    MnSO.sub.4 H.sub.2 O  1.69    ZnSO.sub.4 7H.sub.2 O 0.86    CuSO.sub.4 5H.sub.2 O 0.0025    MS Halides 100X Stock    CaCl.sub.2 2H.sub.2 O 44.0    KI                    0.083    CoCl.sub.2 6H.sub.2 O 0.00125    KH.sub.2 PO.sub.4     17.0    H.sub.3 BO.sub.3      0.62    Na.sub.2 MoO.sub.4 2H.sub.2 O                          0.025    MS FeEDTA 100X Stock    Na.sub.2 EDTA         3.724    FeSO.sub.4 7H.sub.2 O 2.784    B5 Vitamin Stock    10 g m-inositol    100 mg nicotinic acid    100 mg pyridoxine HCl    1 g thiamine    SB55 (per Liter)    10 mL each MS stocks    1 mL B5 Vitamin stock    0.8 g NH.sub.4 NO.sub.3    3.033 g KNO.sub.3    1 mL 2,4-D (10 mg/mL stock)    60 g sucrose    0.667 g asparagine    pH 5.7    For SBP6- substitute 0.5 mL 2,4-D    SB103 (per Liter)    MS Salts    6% maltose    750 mg MgCl.sub.2    0.2% Gelrite    pH 5.7    SB71-1 (per liter)    B5 salts    1 mL B5 vitamin stock    3% sucrose    750 mg MgCl.sub.2    0.2% gelrite    pH 5.7    ______________________________________

Media for the transformation of Brassica Napus cells and the growth ofagrobacterium described in Example 4 is as follows:

Minimal A Bacterial Growth Medium

Dissolve in distilled water:

10.5 grams potassium phosphate, dibasic

4.5 grams potassium phosphate, monobasic

1.0 gram ammonium sulfate

0.5 gram sodium citrate, dihydrate

Make up to 979 mL with distilled water

Autoclave

Add 20 mL filter-sterilized 10% sucrose

Add 1 mL filter-sterilized 1 M MgSO₄

Brassica Callus Medium BC-28

Per liter:

Murashige and Skoog Minimal Organic Medium (MS salts, 100 mg/Li-inositol, 0.4 mg/L thiamine; GIBCO #510-3118)

30 grams sucrose

18 grams mannitol

1.0 mg/L 2,4-D

0.3 mg/L kinetin

0.6% agarose

pH 5.8

Brassica Regeneration Medium BS-48

Murashige and Skoog Minimal Organic Medium

Gamborg B5 Vitamins (SIGMA #1019)

10 grams glucose

250 mg xylose

600 mg MES

0.4% agarose

pH 5.7

Filter-sterilize and add after autoclaving:

2.0 mg/L zeatin

0.1 mg/L IAA

Brassica Shoot Elongation Medium MSV-1A

Murashige and Skoog Minimal Organic Medium

Gamborg B5 Vitamins

10 grams sucrose

0.6% agarose

pH 5.8

Thioesterase assay:

To assay for the presence of thioesterase activity ¹⁴ C! radiolabledacyl ACP substrates were prepared. Preparation of the substratesrequired the isolation of ACP and ACP synthetase from E. coli and theenzymatic reaction of ¹⁴ C! fatty acid with the ACP protein.

Purification of Acyl Carrier Protein (ACP) from E. coli

To frozen E. coli cell paste, (0.5 kg of 1/2 log phase growth of E. coliB grown on minimal media and obtained from Grain Processing Corp,Muscatine, Iowa.) was added 50 mL of a solution 1M in Tris, 1M inglycine, and 0.25 M in EDTA. Ten mL of 1M MgCl₂ was added and thesuspension was thawed in a water bath at 50° C. As the suspensionapproached 37° C. it was transferred to a 37° C. bath, made to 10 mM in2-mercaptoethanol and 20 mg of DNAse and 50 mg of lysozyme were added.The suspension was stirred for 2 h, then sheared by three 20 secondbursts in a Waring Blendor. The volume was adjusted to 1 L and themixture was centrifuged at 24,000×g for 30 min. The resultantsupernatant was centrifuged at 90,000×g for 2 h. The resultanthigh-speed pellet was saved for extraction of acyl-ACP synthase (seebelow) and the supernatant was adjusted to pH 6.1 by the addition ofacetic acid. The extract was then made to 50% in 2-propanol by the slowaddition of cold 2-propanol to the stirred solution at 0° C. Theresulting precipitate was allowed to settle for 2 h and then removed bycentrifugation at 16,000×g. The resultant supernatant was adjusted to pH6.8 with KOH and applied at 2 mL/min to a 4.4×12 cm column ofDEAE-Sephacel which had been equilibrated in 10 mM MES, pH 6.8. Thecolumn was washed with 10 mM MES, pH 6.8 and eluted with 1 L of agradient of LiCl from 0 to 1.7M in the same buffer. Twenty mL fractionswere collected and the location of eluted ACP was determined by applying10 μL of every second fraction to a lane of a native polyacrylamide (20%acrylamide) gel electrophoresis (PAGE). Fractions eluting at about 0.7MLiCl contained nearly pure ACP and were combined, dialyzed overnightagainst water and then lyophilized.

Purification of Acyl-ACP Synthase

Membrane pellets resulting from the high-speed centrifugation describedabove were homogenized in 380 mL of 50 mM Tris-Cl, pH 8.0, and 0.5 M inNaCl and then centrifuged at 80,000×g for 90 min. The resultantsupernatant was discarded and the pellets resuspended in 50 mM Tris-Cl,pH 8.0, to a protein concentration of 12 mg/mL. The membrane suspensionwas made to 2% in Triton X-100 and 10 mM in MgCl₂, and stirred at 0° C.for 20 min before centrifugation at 80,000×g for 90 min. The protein inthe resultant supernatant was diluted to 5 mg/mL with 2% Triton X-100 in50 mM Tris-Cl, pH 8.0 and, then, made to 5 mM ATP by the addition ofsolid ATP (disodium salt) along with an equimolar amount of NaHCO₃. Thesolution was warmed in a 55° C. bath until the internal temperaturereached 53° C. and was then maintained at between 53° C. and 55° C. for5 min. After 5 min the solution was rapidly cooled on ice andcentrifuged at 15,000×g for 15 min. The supernatant from the heattreatment step was loaded directly onto a column of 7 mL Blue Sepharose4B which had been equilibrated in 50 mM Tris-Cl, pH 8.0, and 2% TritonX-100. The column was washed with 5 volumes of the loading buffer, then5 volumes of 0.6 M NaCl in the same buffer and the activity was elutedwith 0.5 M KSCN in the same buffer. Active fractions were assayed forthe synthesis of acyl-ACP, as described below, combined, and bound to 3mL settled-volume of hydroxlyapatite equilibrated in 50 mM Tris-Cl, pH8.0, 2% Triton X-100. The hydroxylapatite was collected bycentrifugation, washed twice with 20 mL of 50 mM Tris-Cl, pH 8.0, 2%Triton X-100. The activity was eluted with two 5 mL washes of 0.5 Mpotassium phosphate, pH 7.5, 2% Triton X-100. The first wash contained66% of the activity and it was concentrated with a 30 kD membranefiltration concentrator (Amicon) to 1.5 mL.

Synthesis of Radiolabeled Acyl-ACP

A solutions of ¹⁴ C! palmitic acid, ¹⁴ C! stearic acid, ¹⁴ C! oleicacid, ¹⁴ C! lauric acid, and ¹⁴ C! decanoic acid (120 nmoles each)prepared in methanol were dried in glass reaction vials. The ACPpreparation described above (1.15 mL, 32 nmoles) was added along with0.1 mL of 0.1 M ATP, 0.05 mL of 80 mM DTT, 0.1 mL of 8 M LiCl, and 0.2mL of 13% Triton X-100 in 0.5 M Tris-Cl, pH 8.0, with 0.1 M MgCl₂. Thereaction was mixed thoroughly and 0.3 mL of the acyl-ACP synthasepreparation was added and the reaction was incubated at 37° C. Afterone-half h intervals a 10 μL aliquot was taken and dried on a smallfilter paper disc. The disc was washed extensively withchloroform:methanol:acetic acid (8:2:1, v:v:v) and radioactivityretained on the disc was taken as a measure of ¹⁴ C!- acyl-ACP. At 2 habout 88% of the ACP had been consumed. The reaction mixes were diluted1 to 4 with 20 mM Tris-Cl, pH 8.0, and applied to 1 mL DEAE-Sephacelcolumns equilibrated in the same buffer. The columns were washed insequence with 5 mL of 20 mM Tris-Cl, pH 8.0, 5 mL of 80% 2-propanol in20 mM Tris-Cl, pH 8.0, and eluted with 0.5 M LiCl in 20 mM Tris-Cl, pH8.0. The column eluates were passed directly onto 3 mL columns ofoctyl-sepharose CL-4B which were washed with 10 mL of 20 mM potassiumphosphate, pH 6.8, and then eluted with 35% 2-propanol in 2 mM potassiumphosphate, pH 6.8. The eluted products were lyophilized and redissolvedat a concentration of 24 μM.

Example 1 ISOLATION OF CDNA'S FOR SOYBEAN AND CANOLA SEED PALMITOYL-ACPTHIOESTERASE

PCR Synthesis of a DNA Probe for an Arabidopsis cDNA with SequenceHomology to a Medium Chain Fatty acyl-ACP Thioesterase

A portion of the sequence of an Arabidopsis cDNA sequenced in theArabidopsis thaliana transcribed genome sequencing project (cloneYAP140T7) obtained from Genbank entry Z17678 (Arabidopsis thalianasystematic cDNA sequencing reveals a gene with homology withUmbellularia californica C12:0-ACP thioesterase. (Francoise et al.,Plant Physiol. Biochem. 31, 599, (1993)) and additional sequence from anArabidopis thaliana cDNA clone obtained using that sequence andcommunicated by Dr. John Ohrolgge (Michigan State University) were usedto make two PCR primers shown in SEQ ID NO:3 (the 5' extending primer)and SEQ ID NO:4 (the 3' extending primer). Total RNA was extracted fromgreen seliques of Arabidopis plants and polysomal RNA was isolatedfollowing the procedure of Kamalay et al., (Cell (1980) 19:935-946). Thepolyadenylated mRNA fraction was obtained by affinity chromatography onoligo-dT cellulose (Aviv et al., Proc. Natl. Acad. Sci. USA (1972)69:1408-1411). Thirteen ng of the polyadenylated mRNA was used astemplate for amplification from oligo-dT using a GeneAmp® RNA-PCR kit(Perkin Elmer Cetus, part number N808-0017). PCR was done at anannealing temperature of 52° C. for 35 cycles. A DNA fragment of about560 base pairs was generated and isolated by agarose gel purification.

The isolated fragment was used as the template for random primerlabeling with ³² P!dCTP.

Cloning of a Brassica napus Seed cDNA Homologus to the ArabidopisThioesterase Like Fragment

The radiolabelled probe was used to screen a Brassica napus seed cDNAlibrary. In order to construct the library, Brassica napus seeds wereharvested 20-21 days after pollination, placed in liquid nitrogen, andpolysomal RNA was isolated following the procedure of Kamalay et al.,(Cell (1980) 19:935-946). The polyadenylated mRNA fraction was obtainedby affinity chromatography on oligo-dT cellulose (Aviv et al., supra).Four micrograms of this mRNA were used to construct a seed cDNA libraryin lambda phage (Uni-ZAP₋₋ XR vector) using the protocol described inthe ZAP-cDNA₋₋ Synthesis Kit (1991 Stratagene Catalog, Item #200400).Approximately 240,000 clones were screened for positively hybridizingplaques using the radiolabelled, PCR derived probe described aboveessentially as described in Sambrook et al., supra except that lowstringency hybridization conditions (50 mM Tris, pH 7.6, 6×SSC,5×Denhardt's, 0.5% SDS, 100 μg denatured calf thymus DNA and 50° C.)were used and post-hybridization washes were performed twice with 2×SSC,0.5% SDS at room temperature for 15 min, then twice with 0.2×SSC, 0.5%SDS at room temperature for 15 min, and then twice with 0.2×SSC, 0.5%SDS at 50° C. for 15 min. Nine positive plaques showing stronghybridization were picked, plated out, and the screening procedure wasrepeated. From the secondary screen four, pure phage plagues wereisolated. Plasmid clones containing the cDNA inserts were obtainedthrough the use of a helper phage according to the in vivo excisionprotocol provided by Stratagene. Double-stranded DNA was prepared usingthe Magic® Miniprep (Promega) and the manufacturers instructions, andthe resulting plasmids were size-analyzed by electrophoresis in agarosegels. One of the four clones, designated p5a, contained an approximately1.5 kb insert which was sequenced from both strands by the di-deoxymethod. The sequence of 1483 bases of the cDNA insert of p5a is shown inSEQ ID NO:2. A second clone, designated p2a was also sequenced and foundto contain a 1673 base pair cDNA shown in SEQ ID NO:31. The sequences ofthe two cDNA inserts are 85% identical overall, they encode peptidesthat are 92% identical overall but which are 94% identical within theregion of the putative mature peptide (the peptide after removal of theplastid transit sequence). The cDNA regions of the two cDNAs whichencode the mature peptides are 90.4% identical. The two cDNAs probablyencode two isozymes of the same activity. Based on the length of thetransit peptides for the two sequences, the length of the respectivecDNAs and alignments to the soybean sequences shown below, it appearsthat the cDNA in clone p5a is a slightly truncated version of the actualmessage while clone p2a represents a full length message. The cDNAisolated from clone p2a has been sequenced and the sequence is given inSEQ ID NO 31.

Cloning of a Soybean Seed cDNA Homologus to the Arabidopis ThioesteraseLike Fragment

A cDNA library was made as follows: Soybean embryos (ca. 50 mg freshweight each) were removed from the pods and frozen in liquid nitrogen.The frozen embryos were ground to a fine powder in the presence ofliquid nitrogen and then extracted by Polytron homogenization andfractionated to enrich for total RNA by the method of Chirgwin et al.(Biochemistry (1979) 18:5294-5299). The nucleic acid fraction wasenriched for poly A⁺ RNA by passing total RNA through an oligo-dTcellulose column and eluting the poly A⁺ RNA with salt as described byGoodman et al. (Meth. Enzymol. (1979) 68:75-90). cDNA was synthesizedfrom the purified poly A⁺ RNA using cDNA Synthesis System (BethesdaResearch Laboratory) and the manufacturer's instructions. The resultantdouble-stranded DNA was methylated by Eco RI DNA methylase (Promega)prior to filling-in its ends with T4 DNA polymerase (Bethesda ResearchLaboratory) and blunt-end ligation to phosphorylated Eco RI linkersusing T4 DNA ligase (Pharmacia, Upsalla Sweden). The double-stranded DNAwas digested with Eco RI enzyme, separated from excess linkers bypassage through a gel filtration column (Sepharose CL-4B), and ligatedto lambda ZAP vector (Stratagene, 1109 N. Torrey Pine Rd., LaJollaCalif.) according to manufacturer's instructions. Ligated DNA waspackaged into phage using the Gigapack packaging extract (Stratagene)according to manufacturer's instructions. The resultant cDNA library wasamplified as per Stratagene's instructions and stored at -80° C.

Following the instructions in the Lambda ZAP Cloning Kit Manual(Stratagene), the cDNA phage library was used to infect E. coli BB4cells and a total of approximately 360,000 plaque forming units wereplated onto 6, 150 mm diameter petri plates. Duplicate lifts of theplates were made onto nitrocellulose filters (Schleicher & Schuell). Thefilters were prehybridized in 25 mL of hybridization buffer consistingof 6×SSPE, 5×Denhardt's solution, 0.5% SDS, 5% dextran sulfate and 0.1mg/mL denatured salmon sperm DNA (Sigma Chemical Co.) at 50° C. for 2 h.Radiolabelled probe based on the Arabidopsis PCR product described abovewas added, and allowed to hybridize for 18 h at 50° C. The filters werewashed exactly as described above. Autoradiography of the filtersindicated that there were 9 strongly hybridizing plaques. The 9 plaqueswere subjected to a second round of screening as before.

From the secondary screen three, pure phage plaques were isolated.Plasmid clones containing the cDNA inserts were obtained through the useof a helper phage according to the in vivo excision protocol provided byStratagene. Double-stranded DNA was prepared using the Magic® Miniprep(Promega) and the manufacturers instructions, and the resulting plasmidswere size-analyzed by electrophoresis in agarose gels. One of the fourclones, designated p233b, contained an approximately 1.2 kb insert onestrand of which was partially sequenced by the di-deoxy method. The 311bases of p233b that were sequenced showed a sequence identity of 81.2%in comparison to the Arabidopsis thioesterase like sequence which wasthe basis for the PCR probe. The other two clones isolated from theinital screening appeared to be cDNA concatomers in which the primaryinserts were of a size similar to p233a. Comparison of the sequence atthe 5 prime end of p233a to both the canola sequence and the Arabidopsissequence indicated that p233a is a 5 prime truncated version of theputative thioesterase. The cDNA insert of p233b was removed by digestionwith Eco RI and the insert was purified by agarose gel electrophoresis.The purified insert was used as the template for random primer labelingas described above. Approximately 150,000 plaque forming units of thesoybean seed cDNA library were plated on three plates as described aboveand duplicate nitrocellulose lifts were screened at high stringency(hybridization at 60° C. in 6×SCC, 0.1% SDS for 18 hr, washing at 60° C.in 0.2×SSC, 0.1% SDS twice for 10 min each). Of 18 positive plaquesobtained, one designated pTE11, and containing a 1.5 kB insert waschosen for sequencing by the di-deoxy method. The sequence of the 1688bases in the soybean cDNA insert of pTE11 are shown in SEQ ID 1.

Example 2 EXPRESSION OF THE CATALYTICALLY ACTIVE PROTEIN ENCODED BY THESOYBEAN AND CANOLA cDNA'S HOMOLOGUS TO THE PUTATIVE THIOESTERASE FROMARABIDOPSIS IN E. COLI

Plasmid vectors for the expression of the portions of the soybean andcanola putative thioesterase cDNA's assumed to encode the pro-proteinwere made using the vector pET-3d (described by F. W. Studier, A. H.Rosenberg, J. J. Dunn and J. W. Dubendorff, Methods in Enzymology Vol.185) and the host cell strain BL21(DE3)(pLysE).

The canola clone p5a was digested with Pvu II and Hin DIII to release a1235 base pair fragment which was blunted with DNA polymerase I beforeisolation by agarose gel electrophoresis. Two oligonucletides weresynthesisized which, when annealed together form the following linkersequence:

5'-CATGGAGGAGCAG (SEQ ID NO:3)

3'-CTCCTCGTC (SEQ ID NO:4)

The linkers were ligated to the 1235 base pair fragment which was thenligated into the Nco I digested and calf intestinal phosphatase treatedpET-3d. The ligation mixture was used to transform competentBL21(DE3)(pLyE) cells and twenty ampicillin resistant clonies were usedto inocculate 5 mL liquid cultures. Plasmid DNA was prepared from thecultures and digested with Pvu II, Nco I and Eco RI to determine thepresence of an insert and its orientation with respect to the T7promoter. Only one insert containing plasmid was obtained, and theorientation of the conding region with respect to the promoter wasreversed. The plasmid DNA was digested with Nco I, the insert isolatedand religated into Nco I digested, phosphatase treated pET-3d as above.The ligation mixture was used to transform competent XL-1 cells. Tenisolated colonies were used to inocculate 5 mL liquid cultures andplasmid DNA was isolated. Three clones were determined to be in theforward direction by their Eco RI restriction fragment pattern. Theregion across the cloning site was sequenced and found to place thestart methionine encoded by the linker DNA sequence in frame with theprotein encoded by the canola cDNA to give the deduce amino acidsequence shown in SEQ ID NO:6.

The soybean cDNA containing plamid pTE11 was digested with Sph I and EcoRI, blunted with DNA polymerase I and the resulting 1208 base pairfragment was isolated by agarose gel electrophoresis. The abovedescribed linkers were ligated to the fragment and the product wasligated into the pET-3b vector as described for the canola cDNA fragmentabove. The ligation mixture was used to transform competent XL-1 cellsand ten of the colonies obtained were used to inocculate 5 mL liquidcultures. Plasmid DNA isolated from the cultures was digested with Nco Ito determine the presence of a cDNA insert and with Hpa I and Sph I todetermine the orientation of the insert relative to the T7 promoter. Oneclone with a correctly oriented insert was obtained and used totransform competent BL21(DE3)(pLysE) cells. The deduced amino acidsequence of the expressed protein is shown in SEQ ID NO:7.

Single colonies of the BL21(DE3)(pLysE) strains containing the pET:canola and the soybean cDNA expression vectors were used to inocculate 5mL of 2×YT media containing 50 mg/L ampicillin. The cultures were grownovernight at 37° C., diluted to 0.1 OD at 600 nm with fresh, ampicillincontaining media and re-grown to 1.5 OD at 600 nm at 37° C. Bothcultures were induced by the addition of IPTG to a final concentrationof 1 mM. Cells were harvested by centrifugation three hr afterinduction. A volume of lysis buffer (50 mM HEPES, pH 7.5, 15 mM NaCl,0.5 mM EDTA, 1 mM DTT and 15% glycerol) approximately equal to thepellet volume was added and the cells were resuspended by vortex mixing.A small amount of 2 mm glass beads and 0.2 M PMSF in 2-propanol to afinal concentration of 0.2 mM was added just before sonication. The celllysate was centrifuged in a microfuge to clear and the supernatent ofthe canola cDNA expressing cell line was diluted one to twenty with 50mM Tricine (pH 8.2, 1 mg/mL BSA and 1 mM DTT) to give a lysate proteinconcentration of 1.8 mg/mL. The cell line expressing the soybean cDNAwas similarly diluted one to five to give a lysate protein concentrationof 2.4 mg/mL.

Acyl-ACP Thioesterase Assay

Reagents and substrates for the thioesterase assay are prepared asdescribed above in the the MATERIALS AND METHODS section. Acyl-ACPthioesterase was assayed as described by Mckeon and Stumpf J. Biol.Chem. (1982) 257:12141-121471!. Each of the radiolabeled acyl-ACP's wereadjusted to concentrations ranging from 0.18 μM to 2.06 μM and a volumeof 40 μL with a reaction buffer consisting of 1 mg/mL bovine serumalbumin in CAPS-NaOH buffer (50 mM) at pH 9.5. Reactions were startedwith lysate from E. coli expressing the plant cDNA's for the putativeacyl-ACP thioesterase from either soybean seed or canola seed andincubated for times varying from 12 seconds to 1 min depending upon theactivity of the fraction. Reactions were terminated by the addition of100 μl of a solution of 5% acetic acid in 2-propanol and extracted twicewith 1 mL each of water saturated hexane. Five mL of ScintiVerse Bio HP(Fisher) scintillation fluid was added to the combined extracts andradioactivity in the released fatty acids was determined by scintilationcounting.

Thioesterase assays done on E. coli extracts from cultures which werenot transformed with thioesterase expressing plasmids had specificactivities of about 0.025 nmole/min/mg protein in the palmitoyl-ACP,stearoyl-ACP and oleoyl-ACP assays when the assay was done at 1 μMsubstrate concentration. Since this E. coli background was from 70 to150 fold less than the activity found in the plant thioesteraseexpressing lines, it is ignored in the following data.

Assays were done at 4 substrate concentrations for the soybean enzymeand at a concentration which gave maximal activity for the canolaenzyme. Assays were done such that less than 25% of the availablesubstrate was consumed at each substrate concentration and the substrateconcentration listed in Table 2 is the average concentration during thetime of the reaction.

                  TABLE 2    ______________________________________    Activity of the Soybean and Canola Thioesterases    Against Palmitoly-ACP, Stearoyl-ACP and Oleoyl-ACP                 SPECIFIC ACTIVITY    SUBSTRATE    (nmole/min/mg protein)    ______________________________________    Soybean Thioesterase    Palmitoyl-ACP    0.18 μM   1.17    0.37 μM   1.87    0.74 μM   3.43    1.01 μM   3.61    Stearoyl-ACP    0.18 μM   0.67    0.41 μM   1.08    0.81 μM   1.80    1.62 μM   1.76    Oleoyl-ACP    0.18 μM   0.21    0.41 μM   0.77    1.03 μM   0.86    2.06 μM   0.98    Palmitoyl-ACP*    0.58 μM   17.6    Docecanoly-ACP*    0.54 μM   0.11    Lauroyl-ACP*    0.54 μM   0.07    Canola Thioesterase    Palmitoyl-ACP    1.01 μM   3.33    Stearoyl-ACP    0.81 μM   1.27    Oleoyl-ACP    1.03 μM   1.76    ______________________________________     *Data from a seperate experiment in which the pET:soybean palmitolyl     thioesterase was expressed to a higher level in BL21 (DE3) cells.

The data in Table 2 shows that both the canola and the soybean enzymesare acyl-ACP thioesterases. While neither enzyme has significantactivity toward lauroyl-ACP or decanoly-ACP which is the substrate forthe enzyme that they were initially idenified as homologus to(Arabidopsis thaliana systematic cDNA sequencing reveals a gene withhomology with Umbellularia californica C12:0-ACP thioesterase. FrancoiseGrellet, Richard Cooke, Monique Raynal, Michele Laudie and MichelDelseny, Plant Physiol. Biochem. 1993 31:599-602), both are activeagainst longer acyl chain-ACP's. Both have a preference of between twoand three fold for palmitoyl-ACP over either stearoyl-ACP or oleoyl-ACP.This is in contrast to the known acyl-ACP thioesterases from thesespecies which show a strong substrate preference for oleoyl-ACP WO9211373!. The enzymes thus represent a second class of acyl-ACPthioesterase, present within the same tissues as the oleoyl-ACPthioesterase which have substrate preference for long chain, saturatedacyl-ACP's.

Example 3 REGULATION OF THE EXPRESSION OF PALMITOYL-ACP THIOESTERASE INSOYBEANS

Construction of Vectors for Transformation of Glycine max for ReducedExpression of Palmitoyl-ACP thioesterase in Developing Soybean Seeds

Plasmids containing the antisense G. max palmitoyl-ACP thioesterase cDNAsequence under control of the soybean beta-conglycinin promoter (Beachyet al., EMBO J. (1985) 4:3047-3053), were constructed. The constructionof vectors expressing the soybean delta-12 desaturase antisense cDNAunder the control of these promoters was facilitated by the use ofplasmids pCW109 and pML18, both of which are described in WO 9411516!.

A unique Not I site was introduced into the cloning region between thebeta-conglicinin promoter and the phaseolin 3' end in pCW109 bydigestion with Nco I and Xba I followed by removal of the singlestranded DNA ends with mung bean exonuclease. Not I linkers (New EnglandBiochemical catalog number NEB 1125) were ligated into the linearizedplasmid to produce plasmid pAW35. The single Not I site in pML18 wasdestroyed by digestion with Not I, filling in the single stranded endswith dNTP's and Klenow fragment followed by re-ligation of thelinearized plasmid. The modified pML18 was then digested with Hind IIIand treated with calf intestinal phosphatase.

The beta-conglicinin:Not I:phaseolin expression cassette in pAW35 wasremoved by digestion with Hind III and the 1.79 kB fragment was isolatedby agarose gel electrophoresis. The isolated fragment was ligated intothe modified and linearized pML18 construction described above. A clonewith the desired orientation was identified by digestion with Not I andXba I to release a 1.08 kB fragment indicating that the orientation ofthe beta-conglycinin transcription unit was the same as the selectablemarker transcription unit. The resulting plasmid was given the namepBS19.

PCR amplification primers SOYTE3

(5'-AAGGAAAAAAGCGGCCGCTGACACAATAGCCCTTCT-3') (SEQ ID NO:5) correspondingto bases 1 to 16 of SEQ ID NO:1 with additional bases to provide a Not Irestriction site and sufficient additional bases to allow Not Idigestion and SOYTE4

(5-AAGGAAAAAAGCGGCCGCGATTTACTGCTGCTTTTC-3') (SEQ ID NO:12) correspondingto the reverse complement of bases 1640 to 1657 of SEQ ID NO:1 withadditional bases to provide a Not I restriction site and sufficientadditional bases to allow Not I digestion were synthesiszed. Using theseprimers, pTE11 as template and standard PCR amplification proccedures(Perkin Elmer Cetus, GeneAmp PCR kit), a 1.6 kB fragment of p233b wasamplified and isolated by agarose gel electrophoresis. The fragment wasdigested overnight at 37° C. with Not I, extracted withphenol/chloroform followed by chloroform extraction and ethanolprecipitation. Plasmid pBS19 was digested with Not I, treated with calfintestinal phosphatase and the linearized plasmid was purified byagarose gel electrophoresis. The Not I digested, PCR amplified fragmentof pTE11 described above was ligated into the linearized pBS19 and theligation mixture used to transform competent Xl-1 cells. A clone inwhich the soybean palmitoyl-ACP cDNA was oriented in the antisensedirection with respect to the beta-conglycinin promoter was identifiedby digestion with Hind III. The antisense orientation releases fragmentsof 1.6 and 1.9 kB while the sense orientation releases fragments of 1.15and 2.3 kB. The antisense soybean palmitoyl-ACP thioesterase plasmid wasdesignated pTC3 and the sense oriented plasmid was designated pTC4.

Transformation Of Somatic Soybean Embryo Cultures

Soybean embryogenic suspension cultures were maintained in 35 mL liquidmedia (SB55 or SBP6, MATERIALS AND METHODS) on a rotary shaker, 150 rpm,at 28° C. with mixed fluorescent and incandescent lights on a 16:8 hday/night schedule. Cultures were subcultured every four weeks byinoculating approximately 35 mg of tissue into 35 mL of liquid medium.

Soybean embryogenic suspension cultures were transformed with pTC3 bythe method of particle gun bombardment (see Kline et al. (1987) Nature(London) 327:70). A DuPont Biolistic PDS1000/HE instrument (heliumretrofit) was used for these transformations.

To 50 mL of a 60 mg/mL 1 mm gold particle suspension was added (inorder); 5 uL DNA(1 ug/uL), 20 uL spermidine (0.1 M), and 50 uL CaCl₂(2.5 M). The particle preparation was agitated for 3 min, spun in amicrofuge for 10 sec and the supernatant removed. The DNA-coatedparticles were then washed once in 400 uL 70% ethanol and are suspendedin 40 uL of anhydrous ethanol. The DNA/particle suspension was sonicatedthree times for 1 sec each. Five uL of the DNA-coated gold particleswere then loaded on each macro carrier disk.

Approximately 300-400 mg of a four week old suspension culture wasplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue were normally bombarded. Membranerupture pressure was set at 1000 psi and the chamber was evacuated to avacuum of 28 inches of mercury. The tissue was placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue was placed back into liquid andcultured as described above.

Eleven days post bombardment, the liquid media was exchanged with freshSB55 containing 50 mg/mL hygromycin. The selective media was refreshedweekly. Seven weeks post bombardment, green, transformed tissue wasobserved growing from untransformed, necrotic embryogenic clusters.Isolated green tissue was removed and inoculated into individual flasksto generate new, clonally propagated, transformed embryogenic suspensioncultures. Thus each new line was treated as independent transformationevent. These suspensions can then be maintained as suspensions ofembryos clustered in an immature developmental stage through subcultureor regenerated into whole plants by maturation and germination ofindividual somatic embryos.

Transformed embryogenic clusters were removed from liquid culture andplaced on a solid agar media (SB103, MATERIALS AND METHODS) containingno hormones or antibiotics. Embryos were cultured for four weeks at 26°C. with mixed fluorescent and incandescent lights on a 16:8 h day/nightschedule before analysis.

Analysis Of Transgenic Glycine Max Embryos Containing An AntisensePalmitoyl-ACP Thioesterase Construct

The vector pTC3 containing the soybean palmitoyl-ACP thioesterase cDNA,in the antisense orientation, under the control of the soybeanbeta-conglycinin promoter as described above gave rise to seven matureembryo lines. A culture of the embryo line used for transformation wascarried through culture to mature embryos without transformation orselection to serve as a fatty acid profile control line. Fatty acidanalysis was performed by gas chromatography of the fatty acyl methylesters essentially as described by Browse et al., (Anal. Biochem. (1986)152:141-145) except that 2.5% H₂ SO₄ in methanol was used as themethylation reagent and samples were heated for 1.5 h at 80° C. toeffect the methanolysis of the embryo lipids using single, matureembryos as the tissue source. Nine to ten embryos from each transformedline and 5 embryos from the untransformed control were analyzed and theresults are shown in Table 3.

                  TABLE 3    ______________________________________    Fatty acids in control soybean embryos and in soybean    embryos transformed with a vector expressing the soybean    palmitoyl-ACP thioesterase in the antisense orientation                          FATTY ACID                          AS % OF TOTAL FATTY ACIDS    EMBRYO LINE               EMBRYO NO. 16:0   18:0 18:1 18:2 18:3    ______________________________________    2872 control               1          12.7   4.6  20.8 53.1 7.9    2872 control               2          13.8   3.1  12.0 58.0 12.0    2872 control               3          15.9   3.9  11.2 53.9 13.9    2872 control               4          14.5   2.9  13.9 57.7 9.2    2872 control               5          15.8   4.4  13.4 51.8 12.4    353/3/1    1          6.4    2.1  11.3 63.1 17.0    353/3/1    2          13.3   3.0  14.5 53.9 14.8    353/3/1    3          6.9    2.0  11.2 62.9 16.9    353/3/1    4          12.1   2.8  9.6  55.8 19.6    353/3/1    5          5.8    1.9  12.3 64.1 15.4    353/3/1    6          10.1   2.3  11.8 57.3 17.7    353/3/1    7          3.9    2.0  17.9 64.1 12.0    353/3/1    8          8.2    2.4  11.0 61.1 16.4    353/3/1    9          8.0    2.4  10.5 59.9 18.3    353/3/1    10         5.1    1.9  13.2 66.8 12.8    353/3/2    1          6.3    2.0  12.0 62.2 17.4    353/3/2    2          9.0    2.5  11.1 60.5 16.8    353/3/2    3          8.3    2.1  11.0 60.3 16.4    353/3/2    4          15.1   2.9  10.1 51.8 19.4    353/3/2    5          6.4    2.1  15.5 60.3 15.5    353/3/2    6          16.1   2.9  11.1 53.5 15.9    353/3/2    7          7.6    2.0  10.3 64.5 15.0    353/3/2    8          5.5    2.1  12.1 64.6 15.7    353/3/2    9          15.9   3.0  9.5  51.8 19.1    353/3/2    10         5.8    2.0  12.8 63.7 14.9    353/3/3    1          7.6    2.5  10.9 61.2 15.9    353/3/3    2          5.4    4.1  20.4 40.2 7.9    353/3/3    3          5.2    1.9  12.6 67.2 12.4    353/3/3    4          4.5    2.0  28.8 54.7 9.1    353/3/3    5          6.7    1.8  11.7 62.1 16.1    353/3/3    6          6.0    1.5  10.3 63.2 17.3    353/3/3    7          6.6    2.5  9.4  65.4 15.0    353/3/3    8          13.2   2.9  21.6 49.9 11.6    353/3/3    9          13.4   3.2  16.4 52.5 12.7    357/1/1    1          8.3    2.1  12.3 63.7 12.8    357/1/1    2          11.1   2.8  11.1 59.3 14.2    357/1/1    3          7.5    2.1  14.1 63.1 12.2    357/1/1    4          7.7    2.4  13.8 62.7 12.4    357/1/1    5          14.2   3.0  10.5 58.2 12.7    357/1/1    6          11.8   2.5  11.3 60.7 12.7    357/1/1    7          13.8   3.2  10.1 56.1 14.8    357/1/1    8          6.3    1.6  12.8 65.8 12.4    357/1/1    9          10.5   2.8  11.2 57.5 16.7    357/1/1    10         7.2    1.9  13.8 62.1 14.1    357/1/2    1          3.4    1.6  18.6 64.6 11.8    357/1/2    2          3.7    1.5  19.0 65.1 11.6    357/1/2    3          5.2    1.4  21.6 56.4 15.5    357/1/2    4          3.9    1.5  12.7 69.5 12.4    357/1/2    5          4.9    1.6  12.2 68.3 12.9    357/1/2    6          4.3    2.0  14.3 66.2 13.0    357/1/2    7          10.5   2.5  12.9 57.7 16.2    357/1/2    8          6.4    1.8  24.7 53.4 13.7    357/1/2    9          11.8   2.3  9.0  57.1 19.4    357/1/2    10         3.1    1.4  14.8 62.3 12.1    357/1/3    1          11.5   2.3  9.7  61.5 14.8    357/1/3    2          9.9    2.3  9.5  64.2 14.0    357/1/3    3          12.7   2.9  13.5 57.3 13.5    357/1/3    4          13.9   3.0  14.3 50.1 18.7    357/1/3    5          14.7   3.0  13.0 53.0 16.3    357/1/3    6          11.8   2.4  9.9  58.3 17.7    357/1/3    7          11.3   2.3  10.1 60.6 15.1    357/1/3    8          11.7   2.4  9.9  61.3 14.2    357/1/3    9          14.4   2.5  5.5  63.3 14.3    357/1/3    10         9.6    2.2  18.7 57.0 12.4    357/5/1    1          4.0    1.3  17.7 63.1 13.3    357/5/1    2          3.8    1.3  16.9 65.0 12.4    357/5/1    3          2.9    1.8  17.6 65.4 11.6    357/5/1    4          4.1    1.4  13.6 66.0 14.0    357/5/1    5          2.8    1.8  17.0 67.3 10.9    357/5/1    6          6.3    1.9  14.3 61.2 15.5    357/5/1    7          3.4    1.0  14.9 68.9 11.1    357/5/1    8          4.5    1.5  17.0 62.4 14.0    357/5/1    9          2.9    0.9  14.5 70.5 10.6    357/5/1    10         3.1    1.1  14.9 69.1 11.0    ______________________________________

The average palmitate content of six of the seven transformed lines issignificantly less than that of the control embryo line. In each ofthese six lines, the average stearate content is also less than thecontrol average. This result is expected if the palmitoyl-ACPthioesterase is responsible for the release of all or part of thepalmitate that is incorporated into triacylglyceride and if theantisense construction has reduced the amount of palmitoyl-ACPthioesterase produced. Since the stearate content of the lines isdecreased rather than increased in correspondence with the decreasedpalmitate, the following may be inferred: The capacity to elongatepalmitoyl-ACP to stearoyl-ACP must be sufficient to convert theincreased flux to stearate, and the capacity to desaturate stearoyl-ACPto oleoly-ACP must also be sufficient to convert the increased flux tooleate. These two events lead to a significant decrease in the totalsaturated fatty acids produced in the transformed embryos. It may alsobe inferred that the oleate desaturating capacity is present in excessof the substrate supplied to it since most of the carbon which was notremoved from the ACP synthetic track is found in the linoleate fraction.

This is seen most clearly in a comparison of lines 357/1/3 and 357/5/1.Line 357/1/3 was transformed but shows little or no alteration in fattyacid phenotype while line 357/5/1 is quite uniform among all testedembryos in producing an altered fatty acid phenotype. The averagepalmitic acid content of the lipid in line 357/5/1 is 3.2 fold less thanthat of line 357/1/3 and the average stearic acid content of 357/1/3 is1.8 fold less than that of line 357/5/1. The combined saturated fattyacid decrease is 12.2% of the total fatty acid, and of that 12.2%,nearly all (11.7%) can be accounted for as increased oleate andlinoleate.

Thus, the combined effect is a soybean embryo line with 65% lesssaturated fatty acid and with increased monounsaturated andpolyunsaturated fatty acid.

From this data we conclude that reduction of the amount of palmitoyl-ACPthioesterase expressed in developing soybean seeds will lead to theproduction of soybean oil with reduced saturated fatty acid content. Thevariation in the amount of antisense effect observed between embryos butwithin a transformed line seen in Table 3 is a characteristic of thistransformation system which is explained more fully below. The relationbetween data taken from the immature embryos and seeds from the zygoticembryos produced on plants regenerated from these somatic embryos isdicussed below.

The Fatty Acid Phenotype Resulting From Antisense Or Co-SuppressionInhibition Of Gene Expression In Soybean Somatic Embryos Is PredictiveOf The Fatty Acid Phenotype Of Seeds Of Plants Regenerated From ThoseEmbryos

Mature somatic soybean embryos are a good model for zygotic embryos.While in the globular embryo state in liquid culture, somatic soybeanembryos contain very low amounts of triacylglycerol or storage proteins,typical of maturing, zygotic soybean embryos. At this developmentalstage, the ratio of total triacylglyceride to total polar lipid(phospholipids and glycolipid) is about 1:4, as is typical of zygoticsoybean embryos at the developmental stage from which the somatic embryoculture was initiated. At the globular stage as well, the mRNAs for theprominent seed proteins, alpha' subunit of beta-conglycinin, kunitztrypsin inhibitor 3, and seed lectin are essentially absent. Upontransfer to hormone-free media to allow differentiation to the maturingsomatic embryo state, triacylglycerol becomes the most abundant lipidclass. As well, mRNAs for alpha'-subunit of beta-conglycinin, kunitztrypsin inhibitor 3 and seed lectin become very abundant messages in thetotal mRNA population. On this basis the somatic soybean embryo systembehaves very similarly to maturing zygotic soybean embryos in vivo, andis therefore a good and rapid model system for analyzing the phenotypiceffects of modifying the expression of genes in the fatty acidbiosynthesis pathway.

Most importantly, the model system is also predictive of the fatty acidcomposition of seeds from plants derived from transgenic embryos. Thisis illustrated with two different antisense constructs in two differenttypes of experiment and in a similar co-suppression experiment:

Liquid culture globular embryos transformed with a chimeric geneconsisting of soybean microsomal delta-15 desaturase (experiment 1, WO9311245) or soybean microsomal delta-12 desaturase (experiment 2) inantisense orientation under the control of a seed-specific promoter(beta-conglycinin promoter) gave rise to mature embryos. The fatty acidcontent of mature somatic embryos from lines transformed with vectoronly (control) and the vector containing the antisense chimeric genes aswell as of seeds of plants regenerated from them was determined. Inexperiment 1, one set of embryos from each line was analyzed for fattyacid content and another set of embryos from that same line wasregenerated into plants. In experiment 2, different lines, containingthe same antisense construct, were used for fatty acid analysis insomatic embryos and for regeneration into plants. In experiment 1, inall cases where a reduced 18:3 content was seen in a transgenic embryoline, compared with the control, a reduced 18:3 content was alsoobserved in segregating seeds of plants derived from that line, whencompared with the control seed (Table 4).

In experiment 2, about 55% of the transformed embryo lines showed anincreased 18:1 content when compared with control lines (Table 5).Soybean seeds, of plants regenerated from different somatic embryo linescontaining the same antisense construct, had a similar frequency (53%)of high oleate transformants as the somatic embryos (Table 5). Onoccasion, an embryo line may be chimeric. That is, 10-70% of the embryoin a line may not contain the transgene. The remaining embryos which docontain the transgene, have been found in all cases to be clonal. Insuch a case, plants with both wild type and transgenic phenotypes may beregenerated from a single, transgenic line, even if most of the embryosanalyzed from that line had a transgenic phenotype. An example of thisis shown in Table 6 in which, of 5 plants regenerated from a singleembryo line, 3 have a high oleic phenotype and two were wild type. Inmost cases, all the plants regenerated from a single transgenic linewill have seeds containing the transgene.

                  TABLE 4    ______________________________________    Percent 18:3 Content of Embryos And Seeds Of    Control and Delta-15 Antisense Construct    Transgenic Soybean Lines    Transformant   Embyro average                               Seed average*    Line           (SD n = 10) (SD, n = 10)    ______________________________________    Control        12.1 (2.6)  8.9 (0.8)    Δ15 antisense, line 1                   5.6 (1.2)   4.3 (1.6)    Δ15 antisense, line 2                   8.9 (2.2)   2.5 (1.8)    Δ15 antisense, line 3                   7.3 (1.1)   4.9 (1.9)    Δ15 antisense, line 4                   7.0 (1.9)   2.4 (1.7)    Δ15 antisense, line 5                   8.5 (1.9)   4.5 (2.2)    Δ15 antisense, line 6                   7.6 (1.6)   4.6 (1.6)    ______________________________________     * Seeds which were segregating with wildtype phenotype and without a copy     of the transgene are not included in these averages

                  TABLE 5    ______________________________________    Oleate Levels in Somatic Embryos And Seeds Of    Regenerated Soybeans Transformed With or Without    Delta-12 Desaturase Antisense Construct                          # of lines Average#    Vector     # of lines with high 18:1                                     % 18:1    ______________________________________    Somatic embryos:    Control    19         0          12.0    D 12 antisense               20         11         35.3    Seeds of regenerated plants:    Control    6          0          18.2    D 12 antisense               17         9          44.4    ______________________________________     *average 18:1 of transgenics is the average of all embryos or seeds     transformed with the delta12 antisense construct in which at least one     embryo or seed from that line had an 18:1 content greater that 2 standard     deviations from the control value (12.0 in embryos, 18.2 in seeds). The     control average is the average of embryos or seeds which do not contain     any transgenic DNA but have been treated in an identical manner to the     transgenics

                  TABLE 6    ______________________________________    Mean of 15-20 seeds from 5 different plants    regenerated from a single embryo line.    Only plants #2, 9 and 11 have seeds with    a high 1B:1 phenotype    Line 4 Plant #                Average seed 18:1%                             Highest seed 18:1%    ______________________________________    1           18.0         26.3    2           33.6         72.1    7           13.6         21.2    9           32.9         57.3    11          24.5         41.7    ______________________________________

In a similar experiment, 75% of the coding region (begininng at the 5'end) of the delta-12 desaturase sequence and of the delta-15 desaturasesequence were each placed behind the b-conglycinin promoter in a singleconstruction for soybean transformation as described above. As inexperiment 2 above, seperate embryo sets were used for analysis at theembryo stage and regeneration into fertile plants. The average 18:1 and18:3 content in five embryos from each of 7 transformed lines is givenin Table 7. Of the 7 lines two clearly have elevated levels of 18:1 aswould be expected of embryos in which the conversion of 18:1 to 18:2 bydelta-12 desaturase is limited due to decreased expression of theenzyme. In these same lines there is a slight decrease in the 18:3content, indicative of a decreased delta-15 desaturase activity.

                  TABLE 7    ______________________________________    The 18:1 and 18:3 content in somatic embryos from seven    lines transfromed with a combined Delta-12 and Delta-15    co-suppression construct.    Values are the mean of five individual embryos    Line            %18:1   %18:3    ______________________________________    561/1/1         45.1    10.1    561/1/2         18.4    13.8    561/1/3         10.7    15.2    561/4/1         39.3    13.4    561/4/2         18.7    13.2    561/4/4         19.7    14.1    561/4/5         14.6    16.1    561/4/6         43.9    12.9    ______________________________________

Twenty, fertile soybean plants were regenerated from somatic embryostransformed with the combined D12/D15 desaturase co-suppressionconstruction described above. Five single seeds from each plant wereanalyzed and of the twenty lines, two showed bulk fatty acid profileswhich suggested that both the D 12 and D 15 desaturase activities weredecreased. The first seeds from transformed plants should be geneticallysegregating for the transgene so single seeds from these two lines wereanalyzed to derive an estimate of the number of transgene locicontributing to the fatty acid phenotype. Ninty nine seeds of line557-2-8-1 were analyzed and 137 seeds of line 557-2-8-2 were analyzed.The fatty acid profile classes from both lines were consistent with twotransgenic loci contributing to the phenotype. The average fatty acidprofile of the seeds which were judged to be in the high segregant classare given in Table 8 for both of these lines.

Table 8

The average fatty acid profiles (as % of total fatty acids) for theprobable double homozygous seeds from two lines segregating forco-suppression transgenes for the Δ12 and Δ15 desaturases. The data arethe mean of 10 single seed profiles for line 557-2-8-1 and 13 singleseed profiles for line 557-2-8-2. The profile from a non-transformedline grown along with the transformed lines in shown for comparison.

    ______________________________________    Line        16:0     18:0   18:1   18:2 18:3    ______________________________________    557-2-8-1   8.6      2.1    82.5   2.5  4.2    557-2-8-2   8.3      2.1    82.0   2.2  5.0    non-transformed                13.3     2.4    17.4   52.3 19.2    ______________________________________

As with the antisense constructions, the fatty acid profiles observed inthe somatic embryos is predictive of the type and magnitude ofalteration in fatty acid profile which will be obtained from the seedsof fertile plants transformed with the same construction as the somaticembryos. Thus, we conclude that an altered fatty acid phenotype observedin a transgenic, mature somatic embryo line is predictive of an alteredfatty acid composition of seeds of plants derived from that line.

Analysis Of Transgenic Glycine Max Embryos Containing A Palmitoyl-ACPThioesterase Construct In The Sense Orientation

The vector pTC4 containg the soybean palmitoyl-ACP thioesterase cDNA, inthe sense orientation, under the control of the soybean beta-conglycininpromoter as described above gave rise to six mature embryo lines in thesoybean somatic embryo system. From 6 to 10 embryos from each of theselines were analyzed for relative content of each fatty acid as describedabove. The results are shown in Table 9.

                  TABLE 9    ______________________________________    Fatty acids in soybean embryos    transformed with a vector expressing the soybean    palmitoyl-ACP thioesterase in the sense orientation                          FATTY ACID                          AS % OF TOTAL FATTY ACIDS    EMBRYO LINE               EMBRYO NO. 16:0   18:0 18:1 18:2 18:3    ______________________________________    361/1/1    1          14.8   3.3  10.9 54.9 14.5    361/1/1    2          13.1   2.7  10.2 56.9 16.3    361/1/1    3          11.7   3.0  14.5 57.4 12.4    361/1/1    4          10.0   3.1  24.1 50.4 11.6    361/1/1    5          10.9   2.6  17.9 54.6 12.9    361/1/1    6          10.5   3.1  27.5 47.3 10.6    361/1/1    7          9.8    3.4  31.5 43.9 10.5    361/1/1    8          10.5   3.4  23.7 50.0 11.0    361/1/1    9          15.0   3.5  9.6  57.5 13.4    361/1/1    10         12.8   3.1  18.7 52.6 12.0    361/1/2    1          3.9    2.3  16.1 66.7 10.1    361/1/2    2          10.2   3.3  26.4 47.5 11.7    361/1/2    3          4.7    2.3  20.8 60.0 11.4    361/1/2    4          3.7    2.5  27.0 56.9 8.8    361/1/2    5          3.9    3.1  37.7 45.8 8.4    361/1/2    6          3.8    2.0  16.6 67.2 9.4    361/2/1    1          13.1   2.9  10.8 55.8 16.7    361/2/1    2          12.0   2.5  11.2 57.3 16.2    361/2/1    3          13.5   3.0  13.2 55.2 3.6    361/2/1    4          13.5   2.8  11.6 56.4 14.9    361/2/1    5          15.3   3.0  7.0  56.9 17.0    361/2/1    6          13.1   2.2  10.1 59.0 14.1    361/2/1    7          13.4   2.9  12.5 56.9 13.6    361/2/1    8          15.1   4.0  13.9 49.4 16.5    361/2/1    9          15.7   3.3  11.2 54.6 13.8    361/2/1    10         13.1   2.7  11.5 58.0 13.8    361/2/2    1          4.4    1.5  40.3 40.9 12.9    361/2/2    2          29.2   3.6  12.8 42.2 11.2    361/2/2    3          2.4    1.0  37.1 45.0 14.4    361/2/2    4          1.7    0.7  46.6 37.3 14.4    361/2/2    5          3.4    1.5  31.2 51.6 12.4    361/2/2    6          4.1    1.4  29.6 46.2 20.1    361/2/2    7          3.7    1.2  37.8 40.1 18.4    361/2/2    8          3.6    1.5  35.4 46.2 13.3    361/2/2    9          5.6    2.4  41.1 31.7 17.6    361/5/1    1          13.7   2.5  11.8 57.8 13.4    361/5/1    2          27.2   3.6  9.8  46.3 11.8    361/5/1    3          16.8   2.8  12.8 53.4 13.4    361/5/1    4          14.6   2.5  11.4 56.6 14.2    361/5/1    5          25.9   4.0  13.8 42.9 12.5    361/5/1    6          25.1   3.3  10.3 49.3 11.0    361/5/1    7          27.2   3.0  4.9  48.6 15.6    361/5/1    8          27.0   3.8  9.8  44.9 13.1    361/5/1    9          28.5   3.5  10.1 45.8 11.2    361/5/1    10         22.8   4.1  14.0 46.1 11.9    361/5/2    1          28.7   3.5  9.8  44.3 12.7    361/5/2    2          31.0   3.5  8.7  43.5 12.4    361/5/2    3          20.2   3.7  9.8  51.0 14.2    361/5/2    4          26.6   3.4  12.9 44.2 11.8    361/5/2    5          27.3   3.5  9.3  44.4 12.4    361/5/2    6          25.9   3.5  11.6 45.2 12.7    361/5/2    7          25.6   3.7  9.2  46.5 13.8    361/5/2    8          25.3   3.7  11.2 46.5 12.3    361/5/2    9          24.8   3.8  9.6  46.4 14.5    361/5/2    10         26.6   3.7  9.8  44.9 14.0    ______________________________________

As is often the case when increasing the expression of an mRNA which isendogenous to the targeted tissue, the effects of both over-expressionof the resulting enzyme and under expresssion of the enzyme due toco-supression are seen in this experiment. While lines 361/1/1 and361/2/1 have fatty acid profiles very similar to control lines (shown inTable 9), most of the embryos in line 361/1/2 have levels of palmiticacid which are about 3 fold lower than controls or transformed lineswhich do not show altered fatty acid phenotype. In contrast, thepalmitic acid content of all of the embryos in line 361/5/2 is increasedand the average palmitic acid content is 26.2% or 1.8 times the averagecontrol embryo. Line 361/2/2 contains 8 embryos which show theco-supression phenotype (low palmitic acid) and one embryo which showsthe over expression phenotype (high palmitic acid content).

In this experiment the effects of altered expression of the soybeanpalmitoyl-ACP thioesterase are seen in both directions, and theresulting phenotypes are as expected from the substrate specificity ofthe enzyme. Modulation of expression upward increases the relativepalmitic acid content and downward decreases the relative palmitic acidcontent.

Example 4 REGULATION OF EXPRESSION OF PALMITOYL-ACP THIOESTERASE INCANOLA

Construction Of Vectors For Transformation Of Brassica Napus For ReducedExpression Of Palmitoyl-ACP thioesterase In Developing Canola Seeds

An extended poly A tail was removed from the canola palmitoyl-ACPthioesterase sequence contained in plasmid p5b as follows. Plasmid p5bwas digested with Eco RI and Ssp I and the 1.5 kB fragment released fromthe pBluescript vector was isolated by agarose gel electrophoresis. Thesingle stranded ends were filled in with Klenow fragment and dNTP's.

Canola napin promoter expression cassettes were constructed as follows:Eight oligonucleotide primers were synthesized based upon the nucleotidesequence of napin lambda clone CGN1-2 published in European Patent 255378. The oligonucleotide sequences were:

BR42: 5'-AACATCAATGGCAGCAACTGCGGA-3' SEQ ID NO:13

BR43: 5'-GCCGGCTGGATTTGTGGCATCAT-3' SEQ ID NO:14

BR45: 5'-CTAGATCTCCATGGGTGTATGTTCTGTAGTGATG-3' SEQ ID NO:15

BR46: 5'-TCAGGCCTGTCGACCTGCGGATCAAGCAGCTTTCA-3' SEQ ID NO:16

BR47: 5'-CTAGATCTGGTACCTAGATTCCAAACGAAIATCCT-3' SEQ ID NO:17

BR48: 5'-AACATCAGGCAAGTTAGCATTTGC-3' SEQ ID NO:18

BR49: 5'-TCAGGCCTGTCGACGAGGTCCTTCGTCAGCATAT-3' SEQ ID NO:19

BR50: 5'-AACGAACCAATGACTTCACTGGGA-3' SEQ ID NO:20

Genomic DNA from the canola variety `Hyola401` (Zeneca Seeds) was usedas a template for PCR amplification of the napin promoter and napinterminator regions. The promoter was first amplified using primers BR42and BR43, and reamplified using primers BR45 and BR46. Plasmid plMC01was derived by digestion of the 1.0 kb promoter PCR product withSalI/BglII and ligation into SalI/BamHI digested pBluescript SK+(Stratagene). The napin terminator region was amplified using primersBR48 and BR50, and reamplified using primers BR47 and BR49. PlasmidplMC06 was derived by digestion of the 1.2 kb terminator PCR productwith SalI/BglII and ligation into SalI/BglII digested pSP72 (Promega).Using plMC06 as a template, the terminator region was reamplified by PCRusing primer

BR57 5'-CCATGGGAGCTCGTCGACGAGGTCCTTCGTCACGAT-3' SEQ ID NO:21 and primer

BR58 5'-GAGCTCCCATGGAGATCTGGTACCTAGATTCCAAAC-3' SEQ ID NO:22

Plasmid plMC101 containing both the napin promoter and terminator wasgenerated by digestion of the PCR product with Sacl/Ncol and ligationinto Sacl/Ncol digested plMC01. Plasmid plMC101 contains a 2.2 kb napinexpression cassette including complete napin 5' and 3' non-translatedsequences and an introduced Ncol site at the translation start ATG.Primer

BR61 5'-GACTATGTTCTGAATTCTCA-3' SEQ ID NO:23 and primer

BR62 5'-GACAAGATCTGCGGCCGCTAAAGAGTGAAGCCGAGGCTC-3' SEQ ID NO:24

were used to PCR amplify an ˜270 bp fragment from the 3' end of thenapin promoter. Plasmid plMC401 was obtained by digestion of theresultant PCR product with EcoRI/BglII and ligation into EcoRI/BglIIdigested plMC 1 01. Plasmid plMC40 1 contains a 2.2 kb napin expressioncassette lacking the napin 5' non-translated sequence and includes aNotl site at the transcription start.

The oligonucleotide sequences were:

BR42 and BR43 corresponding to bases 29 to 52 (BR42) and the complementof bases 1146 to 1169 (BR43) of SEQ ID NO:8.

BR45 and BR46 corresponding to bases 46 to 66 (BR46) and the complementof bases 1028 to 1047 (BR45) of SEQ ID NO:8. In addition BR46 had basescorresponding to a Sal I site (5'-GTCGAC-3') and a few additional bases(5'-TCAGGCCT-3') at its 5' end and BR45 had bases corresponding to a BglII site (5'-AGATCT-3') and two (5'-CT-3') additional bases at the 5' endof the primer.

BR47 and BR48 corresponding to bases 81 to 102 (BR47) and bases 22 to 45(BR48) of SEQ ID NO:10. In addition, BR47 had two (5'-CT-3') additionalbases at the 5' end of the primer followed by bases corresponding to aBgl II site (5'-AGATCT-3') followed by a few additional bases(5'-TCAGGCCT-3'),

BR49 and BR50 corresponding to the complement of bases 1256 to 1275(BR49) and the complement of bases 1274 to 1297 (BR50) of SEQ ID NO:10.In addition BR49 had bases corresponding to a Sal I site (5'-GTCGAC-3')and a few additional bases (5'-TCAGGCCT-3') at its 5' end.

BR57 and BR58 corresponding to the complement of bases 1258 to 1275(BR57) and bases 81 to 93 (BR58) of SEQ ID NO:10. In addition the 5' endof BR57 had some extra bases (5'-CCATGG-3') followed by basescorresponding to a Sac I site (5'-GAGCTC-3') followed by more additionalbases (5'-GTCGACGAGG-3') (SEQ ID NO:25). The 5' end of BR58 hadadditional bases (5'-GAGCTC-3') followed by bases corresponding to a NcoI site (5'-CCATGG-3') followed by additional bases (5' AGATCTGGTACC-3')(SEQ ID NO:26).

BR61 and BR62 corresponding to bases 745 to 764 (BR61) and bases 993 to1013 (BR62) of SEQ ID NO:8. In addition the 5' end of BR 62 hadadditional bases (5'-GACA-3') followed by bases corresponding to a BglII site (5'-AGATCT-3') followed by a few additional bases(5'-GCGGCCGC-3').

Genomic DNA from the canola variety `Hyola401` (Zeneca Seeds) was usedas a template for PCR amplification of the napin promoter and napinterminator regions. The promoter was first amplified using primers BR42and BR43, and reamplified using primers BR45 and BR46. Plasmid pIMC01was derived by digestion of the 1.0 kb promoter PCR product withSalI/BglII and ligation into SalI/BamHI digested pBluescriptSK+(Stratagene). The napin terminator region was amplified using primersBR48 and BR50, and reamplified using primers BR47 and BR49. PlasmidpIMC06 was derived by digestion of the 1.2 kb terminator PCR productwith SalI/BglII and ligation into SalI/BglII digested pSP72 (Promega).Using pIMC06 as a template, the terminator region was reamplified by PCRusing primer BR57 and primer BR58. Plasmid pIMC101 containing both thenapin promoter and terminator was generated by digestion of the PCRproduct with SacI/NcoI and ligation into SacI/NcoI digested pIMC01.Plasmid pIMC101 contains a 2.2 kb napin expression cassette includingcomplete napin 5' and 3' non-translated sequences and an introduced NcoIsite at the translation start ATG. Primer BR61 and primer BR62 were usedto PCR amplify an ˜270 bp fragment from the 3' end of the napinpromoter. Plasmid pIMC401 was obtained by digestion of the resultant PCRproduct with EcoRI/BglII and ligation into EcoRI/BglII digested pIMC101.Plasmid pIMC401 contains a 2.2 kb napin expression cassette lacking thenapin 5' non-translated sequence and includes a NotI site at thetranscription start.

Plasmid pIMC401 was digested with Not I and the single stranded endsfilled with dNTP's and Klenow fragment. The linearized plasmid wastreated with calf intestinal phosphatase. The phospatase treated andlinearized plasmid was ligated to the blunted, 1.5 kB fragment of canolapalmitoyl-ACP thioesterase described above. Transformation of competentE. coli cells with the ligation mixture resulted in the isolation ofclones in which the plant cDNA sequence was in the sense orientationwith respect to the napin promoter (pIMC29) and in the antisenseorientation (pIMC30).

The vector for transformation of the antisense palmitoyl-ACPthioesterase construction under control of the napin promoter intoplants using Agrobacterium tumefaciens was produced by constructing abinary Ti plasmid vector system (Bevan, (1984) Nucl. Acids Res.12:8711-8720). One starting vector for the system, (pZS199) is based ona vector which contains: (1) the chimeric gene nopalinesynthase/neomycin phosphotransferase as a selectable marker fortransformed plant cells (Brevan et al. (1984) Nature 304:184-186), (2)the left and right borders of the T-DNA of the Ti plasmid (Brevan et al.(1984) Nucl. Acids Res. 12:8711-8720), (3) the E. coli lacZα-complementing segment (Vieria and Messing (1982) Gene 19:259-267) withunique restriction endonuclease sites for Eco RI, Kpn I, Bam HI, and SalI, (4) the bacterial replication origin from the Pseudomonas plasmidpVS1 (Itoh et al. (1984) Plasmid 11:206-220), and (5) the bacterialneomycin phosphotransferase gene from Tn5 (Berg et al. (1975) Proc.Natnl. Acad. Sci. U.S.A. 72:3628-3632) as a selectable marker fortransformed A. tumefaciens. The nopaline synthase promoter in the plantselectable marker was replaced by the 35S promoter (Odell et al. (1985)Nature, 313:810-813) by a standard restriction endonuclease digestionand ligation strategy. The 35S promoter is required for efficientBrassica napus transformation as described below.

The binary vectors containing the sense and antisense palmitoyl-ACPthioesterase expression cassettes were constructed by digesting pIMC29and pIMC30 with Sal I to release the napin:palmitoyl-ACP thioesterasecDNA:napin 3' sequence and agarose gel purification of the 3.8 kBfragments. Plasmid pZS199 was also digested with Sal I and the 3.8 kBfragments isolated from pIMC29 and pIMC30 were ligated into thelinearized vector. Transformation and isolation of clones resulted inthe binary vector containing the sense construct (pIMC129) and theantisense construct (pIMC130).

Agrobacterium-Mediated Transformation Of Brassica Napus

The binary vectors pIMC129 and pIMC130 were transferred by a freeze/thawmethod (Holsters et al. (1978) Mol. Gen. Genet. 163:181-187) to theAgrobacterium strain LBA4404/pAL4404 (Hockema et al. (1983), Nature303:179-180).

Brassica napus cultivar "Westar" was transformed by co-cultivation ofseedling pieces with disarmed Agrobacterium tumefaciens strain LBA4404carrying the the appropriate binary vector.

B. napus seeds were sterilized by stirring in 10% Chlorox, 0.1% SDS forthirty min, and then rinsed thoroughly with sterile distilled water. Theseeds were germinated on sterile medium containing 30 mM CaCl₂ and 1.5%agar, and grown for six days in the dark at 24° C.

Liquid cultures of Agrobacterium for plant transformation were grownovernight at 28° C. in Minimal A medium containing 100 mg/L kanamycin.The bacterial cells were pelleted by centrifugation and resuspended at aconcentration of 10⁸ cells/mL in liquid Murashige and Skoog MinimalOrganic medium containing 100 μM acetosyringone.

B. napus seedling hypocotyls were cut into 5 mm segments which wereimmediately placed into the bacterial suspension. After 30 min, thehypocotyl pieces were removed from the bacterial suspension and placedonto BC-28 callus medium containing 100 μM acetosyringone. The planttissue and Agrobacteria were co-cultivated for three days at 24° C. indim light.

The co-cultivation was terminated by transferring the hypocotyl piecesto BC-28 callus medium containing 200 mg/L carbenicillin to kill theAgrobacteria, and 25 mg/L kanamycin to select for transformed plant cellgrowth. The seedling pieces were incubated on this medium for threeweeks at 24° C. under continuous light.

After three weeks, the segments were transferred to BS-48 regenerationmedium containing 200 mg/L carbenicillin and 25 mg/L kanamycin. Planttissue were subcultured every two weeks onto fresh selectiveregeneration medium, under the same culture conditions described for thecallus medium. Putatively transformed calli grow rapidly on regenerationmedium; as calli reach a diameter of about 2 mm, they are removed fromthe hypocotyl pieces and placed on the same medium lacking kanamycin.

Shoots begin to appear within several weeks after transfer to BS-48regeneration medium. As soon as the shoots form discernable stems, theyare excised from the calli, transferred to MSV-1A elongation medium, andmoved to a 16:8-h photoperiod at 24° C.

Once shoots have elongated several internodes, they are cut above theagar surface and the cut ends are dipped in Rootone. Treated shoots areplanted directly into wet Metro-Mix 350 soiless potting medium. The potsare covered with plastic bags which are removed when the plants areclearly growing--after about ten days.

Plants are grown under a 16:8-h photoperiod, with a daytime temperatureof 23° C. and a nightime temperature of 17° C. When the primaryflowering stem begins to elongate, it is covered with a meshpollen-containment bag to prevent outcrossing. Self-pollination isfacilitated by shaking the plants several times each day, and seedsmature by about 90 days following transfer to pots.

The relative content of each of the 7 main fatty acids in the seed lipidwas analyzed as follows: Twenty seeds taken at random from a sample of25 pods from each plant were ground in 0.5 mL of 2-propanol. Twenty fiveμL of the resulting extract was transferred to a glass tube and thesolvent evaporated under a nitrogen stream. The dry residue wassubjected to methanolysis in 0.5 mL of 1% sodium methoxide in methanolat 60° C. for 1 hour. The fatty acid methyl esters produced wereextracted into 1 mL of hexane and 0.5 mL of water was added to thesolvent mixture to wash methanol from the hexane layer. A portion of thehexane layer was transferred to a sample vial for analysis by gas-liquidchromatography as described in Example 3 above. While seven fatty acidswere analyzed, only the relative contribution of the 5 main fatty acidsto the total are shown in Tables 10, 11 and 12 below.

                  TABLE 10    ______________________________________    The relative contribution of 5 fatty    acids to the bulk seed fatty acid content in    segregating canola plants transformed with pIMC129    containing the canola palmitoyl-ACP thioesterase    in the sense orientation to the Napin promotor                 FATTY ACID                 AS % OF TOTAL FATTY ACIDS    TRANSFORMANT NO.                 16:0     18:0   18:1   18:2 18:3    ______________________________________    129-511      4.1      1.4    67.9   19.0 5.9    129-186      4.2      1.4    66.5   20.0 5.9    129-230      4.2      1.2    63.9   21.0 7.9    129-258      4.0      1.4    57.2   25.5 10.0    129-107      4.7      1.7    59.0   24.1 8.4    129-457      4.3      1.3    62.0   22.8 7.7    129-381      4.2      1.1    58.0   24.8 10.0    129-515      4.4      1.3    63.4   21.8 7.5    129-122      4.0      1.4    63.0   21.4 8.4    129-176      4.1      1.4    65.7   19.6 7.5    129-939      4.4      1.7    64.8   19.2 8.2    129-303      4.2      1.5    62.3   21.4 9.4    129-208      3.8      1.4    66.9   18.0 8.2    129-835      4.3      1.6    58.0   24.5 9.7    129-659      4.0      1.6    60.8   22.2 10.0    129-44       4.2      1.8    66.0   18.4 7.7    129-756      3.9      1.6    60.0   22.4 10.0    129-30       4.0      1.7    64.8   18.7 9.6    129-340      3.8      1.7    67.1   17.4 7.9    129-272      3.9      1.8    59.4   21.3 12.0    129-358      4.2      1.5    60.7   20.8 11.0    129-223      4.3      1.6    63.4   20.6 8.3    129-314      4.1      2.0    61.8   21.4 9.4    129-657      4.2      1.8    64.8   18.3 9.1    129-151      4.2      1.4    62.5   20.8 9.2    129-40       4.3      1.6    63.8   20.8 7.8    129-805      4.4      2.2    61.6   19.4 10.0    129-44       4.1      1.6    64.2   19.1 8.7    129-288      3.5      1.5    65.1   18.9 8.9    129-833      4.2      1.7    58.8   23.6 9.4    129-889      4.6      2.8    57.6   26.4 9.5    129-247      5.7      1.5    52.8   27.2 13.0    129-355      4.3      2.3    66.0   19.1 6.3    129-631      4.5      2.3    66.7   19.4 5.6    129-73       5.0      2.5    65.4   20.8 6.4    129-407      3.9      1.5    65.4   21.2 6.1    westar       4.0      1.7    64.0   19.7 8.5    ______________________________________

None of the transformed plants analyzed have fatty acid profiles whichare markedly different from that expected in canola seeds. Plants number129-805, 129-889, and 129-73 are slightly elevated in their saturatedfatty acid content and may represent lines with a low amount of overexpression. Since the transformation event gives rise to a plant whichis heterozygous for the introduced transgene, the seed from these plantsis segregating with respect to the transgene copy number. If, asexpected, the fatty acid phenotype is additive with respect to thetransgene copy number, the full effect cannot be seen in bulk seedpopulation until the second generation past transformation. Furtheranalysis will be done on subsequent generations of plants with modestincreases in saturated fatty acid content.

There is no strong evidence for the low palmitate phenotype expectedfrom a co-supressing transformant. In contrast to soybean however,co-supression in canola is a rare transformation event. In ourexperience with other genes in the fatty acid biosynthetic pathway, asmany as 200 transformed lines have been required to observe a strongco-supression phenotype.

                  TABLE 11    ______________________________________    The relative contribution of 5 fatty    acids to the bulk seed fatty acid content in    segregating canola plants transformed with pIMC130    containing the canola palmitoyl-ACP thioesterase    in the antisense orientation to the Napin promotor                 FATTY ACID                 AS % OF TOTAL FATTY ACIDS    TRANSFORMANT NO.                 16:0     18:0   18:1   18:2 18:3    ______________________________________    130-220      4.0      1.7    65.5   20.1 6.4    130-527      4.1      1.7    62.6   19.7 10.0    130-529      4.4      1.7    69.6   17.4 4.6    130-347      4.0      1.4    64.8   21.3 6.1    130-738      4.9      1.5    56.6   27.4 7.3    130-317      4.2      1.4    62.4   22.7 7.6    130-272      4.8      1.6    62.7   23.2 6.4    130-412      4.4      1.4    63.7   22.3 6.7    130-119      3.9      1.1    59.7   25.7 7.9    130-257      5.0      1.8    62.1   20.5 8.8    130-677      4.8      1.2    53.6   28.6 10.0    130-310      4.6      1.6    61.6   23.0 7.3    130-323      4.0      2.0    67.8   16.9 7.4    130-699      4.1      1.1    62.8   23.4 6.8    130-478      5.0      2.0    57.0   23.4 11.0    130-651      4.4      1.6    66.0   19.2 7.7    130-126      3.4      1.7    68.4   16.2 8.6    130-465      5.1      1.9    58.5   24.1 10.0    130-234      4.2      1.6    64.2   20.9 7.8    130-661      4.4      1.4    60.6   22.8 9.6    130-114      4.2      1.4    65.2   19.7 7.8    130-305      4.6      1.6    58.6   23.9 10.0    130-240      4.1      1.4    69.1   17.4 6.5    130-660      4.1      1.4    67.0   18.5 7.2    130-350      4.1      1.5    62.5   21.1 9.8    130-36       4.1      1.9    61.4   21.7 8.9    130-527      4.1      1.5    64.7   19.0 9.0    130-33       4.0      1.1    62.6   22.1 9.1    westar       4.0      1.7    64.0   19.7 8.5    ______________________________________

The average palmitic acid content for the 28 transformants analyzed is4.3 with a standard deviation of the mean of 0.39. While there are nolines which deviate greatly from the mean in bulk seed analysis, line130-126 is in exess of 2 standard deviations lower than the mean. Sincethis could be indicative of a weak antisense phenotype observed in asegregating seed population as described above, 12 single seeds from theplant were analyzed for relative fatty acid content along with 12 singleseeds from a non-transformed Westar plant grown in the same growthchamber and planted at a comparable date. The results of those analysesare shown in Table 12.

                  TABLE 12    ______________________________________    The relative contribution of 5 fatty acids    to total fatty acid content in single seeds    from transformant 130-126 and from single    seeds of a non-transformed oontrol plant                 FATTY ACID                 AS % OF TOTAL FATTY ACIDS    TRANSFORMANT NO.                 16:0     18:0   18:1   18:2 18:3    ______________________________________    130-126      3.07     1.51   67.27  17.26                                             8.74    130-126      3.11     1.74   64.70  18.19                                             9.47    130-126      3.20     1.66   69.71  16.21                                             7.40    130-126      3.47     1.77   69.98  15.66                                             6.73    130-126      3.76     2.04   71.26  15.42                                             5.00    130-126      3.56     1.80   71.74  15.47                                             4.83    130-126      3.30     2.05   65.22  18.11                                             9.37    130-126      3.45     1.91   71.32  14.72                                             5.94    130-126      4.30     1.90   64.97  17.91                                             8.84    130-126      2.95     1.93   65.57  17.27                                             10.30    130-126      3.44     1.71   69.98  16.06                                             6.26    130-126      3.43     1.81   72.40  14.78                                             5.02    WESTAR4/B    3.81     1.71   62.46  20.46                                             9.70    WESTAR4/8    4.28     1.42   63.27  20.86                                             8.30    WESTAR4/8    4.00     1.55   68.80  18.08                                             5.30    WESTAR4/8    4.19     1.97   61.51  20.01                                             10.40    WESTAR4/8    4.37     1.60   63.92  20.02                                             7.96    WESTAR4/8    4.41     1.45   62.95  20.39                                             8.36    WESTAR4/8    4.12     1.84   60.90  21.19                                             10.00    WESTAR4/8    3.89     1.69   63.63  19.68                                             8.99    WESTAR4/8    3.97     1.73   67.68  17.57                                             6.43    WESTAR4/8    3.97     1.78   63.78  19.47                                             8.94    WESTAR4/8    3.85     1.76   64.85  18.56                                             8.65    WESTAR4/8    4.06     1.69   63.74  20.16                                             8.52    ______________________________________

The mean relative palmitic acid content of the 12 seeds fromtransformant 130-126 is 3.42% and the standard deviation of the mean is0.359, while the mean palmitic acid content of the 12 control seeds is4.08 with a standard deviation of the mean of 0.20. The lower mean,greater standard deviation and wider range of observed palmitic acidcontents are all indicative of a segregating population in which theseeds homozygous for the antisense transgene for the canolapalmitoyl-ACP thioesterase produce slightly less palmitic acid. Theobserved phenotype will be confirmed by analysis of bulk seeds frommultiple plants in the next generation.

As stated for the sense construction above, the occurrence of maximallyaltered fatty acid phenotypes are rare transformation events in canola.Thus, the phenotype of the low palmitate segregating seed intransformant 130-126 is indicative that the antisense under expressionof palmitoyl-ACP thioesterase in canola seeds is capable of decreasingthe production of saturated fatty acids but does not indicate theminimum palmitic acid content which may be achieved by this method.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  32    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1688 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:    - ACAATTACAC TGTCTCTCTC TTTTCCAAAA TTAGGGAAAC AACAAGGACG CA - #AAATGACA      60    - CAATAGCCCT TCTTCCCTGT TTCCAGCTTT TCTCCTTCTC TCTCTCTCCA TC - #TTCTTCTT     120    - CTTCTTCACT CAGTCAGATC CAACTCCTCA GATAACACAA GACCAAACCC GC - #TTTTTCTG     180    - CATTTCTAGA CTAGACGTTC TACCGGAGAA GCGACCTTAG AAATTCATTA TG - #GTGGCAAC     240    - AGCTGCTACT TCATCATTTT TCCCTGTTAC TTCACCCTCG CCGGACTCTG GT - #GGAGCAGG     300    - CAGCAAACTT GGTGGTGGGC CTGCAAACCT TGGAGGACTA AAATCCAAAT CT - #GCGTCTTC     360    - TGGTGGCTTG AAGGCAAAGG CGCAAGCCCC TTCGAAAATT AATGGAACCA CA - #GTTGTTAC     420    - ATCTAAAGAA AGCTTCAAGC ATGATGATGA TCTACCTTCG CCTCCCCCCA GA - #ACTTTTAT     480    - CAACCAGTTG CCTGATTGGA GCATGCTTCT TGCTGCTATC ACAACAATTT TC - #TTGGCCGC     540    - TGAAAAGCAG TGGATGATGC TTGATTGGAA GCCACGGCGA CCTGACATGC TT - #ATTGACCC     600    - CTTTGGGATA GGAAAAATTG TTCAGGATGG TCTTGTGTTC CGTGAAAACT TT - #TCTATTAG     660    - ATCATATGAG ATTGGTGCTG ATCGTACCGC ATCTATAGAA ACAGTAATGA AC - #CATTTGCA     720    - AGAAACTGCA CTTAATCATG TTAAAAGTGC TGGGCTTCTT GGTGATGGCT TT - #GGTTCCAC     780    - GCCAGAAATG TGCAAAAAGA ACTTGATATG GGTGGTTACT CGGATGCAGG TT - #GTGGTGGA     840    - ACGCTATCCT ACATGGGGTG ACATAGTTCA AGTGGACACT TGGGTTTCTG GA - #TCAGGGAA     900    - GAATGGTATG CGTCGTGATT GGCTTTTACG TGACTCCAAA ACTGGTGAAA TC - #TTGACAAG     960    - AGCTTCCAGT GTTTGGGTCA TGATGAATAA GCTAACACGG AGGCTGTCTA AA - #ATTCCAGA    1020    - AGAAGTCAGA CAGGAGATAG GATCTTATTT TGTGGATTCT GATCCAATTC TG - #GAAGAGGA    1080    - TAACAGAAAA CTGACTAAAC TTGACGACAA CACAGCGGAT TATATTCGTA CC - #GGTTTAAG    1140    - TCCTAGGTGG AGTGATCTAG ATATCAATCA GCATGTCAAC AATGTGAAGT AC - #ATTGGCTG    1200    - GATTCTGGAG AGTGCTCCAC AGCCAATCTT GGAGAGTCAT GAGCTTTCTT CC - #ATGACTTT    1260    - AGAGTATAGG AGAGAGTGTG GTAGGGACAG TGTGCTGGAT TCCCTGACTG CT - #GTATCTGG    1320    - GGCCGACATG GGCAATCTAG CTCACAGCGG GCATGTTGAG TGCAAGCATT TG - #CTTCGACT    1380    - GGAAAATGGT GCTGAGATTG TGAGGGGCAG GACTGAGTGG AGGCCCAAAC CT - #GTGAACAA    1440    - CTTTGGTGTT GTGAACCAGG TTCCAGCAGA AAGCACCTAA GATTTGAAAT GG - #TTAACGAT    1500    - TGGAGTTGCA TCAGTCTCCT TGCTATGTTT AGACTTATTC TGGTTCCCTG GG - #GAGAGTTT    1560    - TGCTTGTGTC TATCCAATCA ATCTACATGT CTTTAAATAT ATACACCTTC TA - #ATTTGTGA    1620    - TACTTTGGTG GGTAAGGGGG AAAAGCAGCA GTAAATCTCA TTCTCATTGT AA - #TTAAAAAA    1680    #        1688    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1483 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:    - GGCACGAGCT CATTCTTCCC TCTCCCATCT TCCCCACTCG ACCCCACCGC AA - #AAACCAAC      60    - AAAGTCACCA CCTCCACCAA CTTCTCCGGC ATCTTCCCCA CTCCAAACTC CT - #CCGGCAGA     120    - TGAAGGTTAA ACCAAACGCT CAGGCCCCAC CCAAGATCAA CGGCAAGAGA GT - #CGGTCTCC     180    - CTTCTGGCTC GGTGAAGCCT GATAACGAGA CGTCCTCACA GCATCCCGCA GC - #ACCGAGGA     240    - CGTTCATCAA CCAGCTGCCT GACTGGAGCA TGCTTCTTGC TGCAATAACA AC - #CGTCTTCT     300    - TGGCGGCTGA GAAGCAGTGG ATGATGCTTG ACTGGAAACC GAGGCGCTCT GA - #CGTGATTA     360    - TGGATCCGTT TGGGTTAGGG AGGATCGTTC AGGATGGGCT TGTGTTCCGT CA - #GAATTTCT     420    - CTATTCGGTC TTATGAGATA GGTGCTGATC GCTCTGCGTC TATAGAAACG GT - #TATGAATC     480    - ATTTACAGGA AACGGCACTA AACCATGTTA AGACTGCTGG ACTGCTTGGA GA - #TGGGTTTG     540    - GTTCTACTCC TGAGATGGTT AAGAAGAACT TGATTTGGGT TGTTACTCGT AT - #GCAGGTTG     600    - TCGTTGATAA ATATCCTACT TGGGGAGATG TTGTGGAAGT AGATACATGG GT - #GAGCCAGT     660    - CTGGAAAGAA CGGTATGCGT CGTGATTGGC TAGTTCGAGA TGGCAATACT GG - #AGAAATTT     720    - TAACAAGAGC ATCAAGTGTG TGGGTGATGA TGAATAAACT GACAAGAAGA TT - #ATCAAAGA     780    - TTCCTGAAGA GGTTCGAGGG GAGATAGAGC CTTACTTTGT TAATTCTGAC CC - #AGTCCTTG     840    - CCGAGGACAG CAGAAAGTTA ACAAAACTTG ATGACAAGAC TGCTGACTAT GT - #TCGTTCTG     900    - GTCTCACTCC GCGTTGGAGT GACTTGGATG TTAACCAGCA CGTTAACAAT GT - #GAAGTACA     960    - TCGGGTGGAT ACTGGAGAGT GCACCTGTGG GGATGATGGA GAGTCAGAAG CT - #GAAAAGCA    1020    - TGACTCTGGA GTATCGCAGG GAGTGCGGGA GGGACAGTGT GCTTCAGTCC CT - #CACCGCGG    1080    - TTTCGGGCTG CGATATCGGT AGCCTCGGGA CGGCTGGTGA AGTGGAATGT CA - #GCATCTGC    1140    - TCCGTCTCCA GGATGGAGCT GAAGTGGTGA GAGGAAGAAC AGAGTGGAGT TC - #CAAAACAT    1200    - CAACAACAAC TTGGGACATC ACACCGTGAA AAGAATATAG CAAACATGGG TT - #CTTTGGTT    1260    - CGTTTGTAAA ACTATACTAC CTTGCTTGCA ACCACCACTA CTCAAAAACA GT - #TTGGGCCA    1320    - CCTTTGTATA TTTTCTTTGG TTCTTATTTT TTTTCTTCTT GGAGGTCCCT TT - #TTATTATA    1380    - TTTATTTTTT CTTTTGGGTG CCAGACAAAG GCAAATAACT TTCTTATCCT AA - #TATTATTT    1440    #                 148 - #3TTAAAAAA AAAAAAAAAA AAA    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  13 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:    #      13    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH:  9 base              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:    #          9    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  36 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:    #       36         GCTG ACACAATAGC CCTTCT    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  328 ami - #no acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  unkn - #own              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE:  protein    -    (iii) HYPOTHETICAL:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:    - Met Glu Glu Gln Leu Pro Asp Trp Ser Met Le - #u Leu Ala Ala Ile Thr    #                15    - Thr Val Phe Leu Ala Ala Glu Lys Gln Trp Me - #t Met Leu Asp Trp Lys    #            30    - Pro Arg Arg Ser Asp Val Ile Met Asp Pro Ph - #e Gly Leu Gly Arg Ile    #        45    - Val Gln Asp Gly Leu Val Phe Arg Gln Asn Ph - #e Ser Ile Arg Ser Tyr    #    60    - Glu Ile Gly Ala Asp Arg Ser Ala Ser Ile Gl - #u Thr Val Met Asn His    #80    - Leu Gln Glu Thr Ala Leu Asn His Val Lys Th - #r Ala Gly Leu Leu Gly    #                95    - Asp Gly Phe Gly Ser Thr Pro Glu Met Val Ly - #s Lys Asn Leu Ile Trp    #           110    - Val Val Thr Arg Met Gln Val Val Val Asp Ly - #s Tyr Pro Thr Trp Gly    #       125    - Asp Val Val Glu Val Asp Thr Trp Val Ser Gl - #n Ser Gly Lys Asn Gly    #   140    - Met Arg Arg Asp Trp Leu Val Arg Asp Gly As - #n Thr Gly Glu Ile Leu    145                 1 - #50                 1 - #55                 1 -    #60    - Thr Arg Ala Ser Ser Val Trp Val Met Met As - #n Lys Leu Thr Arg Arg    #               175    - Leu Ser Lys Ile Pro Glu Glu Val Arg Gly Gl - #u Ile Glu Pro Tyr Phe    #           190    - Val Asn Ser Asp Pro Val Leu Ala Glu Asp Se - #r Arg Lys Leu Thr Lys    #       205    - Leu Asp Asp Lys Thr Ala Asp Tyr Val Arg Se - #r Gly Leu Thr Pro Arg    #   220    - Trp Ser Asp Leu Asp Val Asn Gln His Val As - #n Asn Val Lys Tyr Ile    225                 2 - #30                 2 - #35                 2 -    #40    - Gly Trp Ile Leu Glu Ser Ala Pro Val Gly Me - #t Met Glu Ser Gln Lys    #               255    - Leu Lys Ser Met Thr Leu Glu Tyr Arg Arg Gl - #u Cys Gly Arg Asp Ser    #           270    - Val Leu Gln Ser Leu Thr Ala Val Ser Gly Cy - #s Asp Ile Gly Ser Leu    #       285    - Gly Thr Ala Gly Glu Val Glu Cys Gln His Le - #u Leu Arg Leu Gln Asp    #   300    - Gly Ala Glu Val Val Arg Gly Arg Thr Glu Tr - #p Ser Ser Lys Thr Ser    305                 3 - #10                 3 - #15                 3 -    #20    - Thr Thr Thr Trp Asp Ile Thr Pro                    325    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  328 ami - #no acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  unkn - #own              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE:  protein    -    (iii) HYPOTHETICAL:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:    - Met Glu Glu Gln Leu Leu Ala Ala Ile Thr Th - #r Ile Phe Leu Ala Ala    #                15    - Glu Lys Gln Trp Met Met Leu Asp Trp Lys Pr - #o Arg Arg Pro Asp Met    #            30    - Leu Ile Asp Pro Phe Gly Ile Gly Lys Ile Va - #l Gln Asp Gly Leu Val    #        45    - Phe Arg Glu Asn Phe Ser Ile Arg Ser Tyr Gl - #u Ile Gly Ala Asp Arg    #    60    - Thr Ala Ser Ile Glu Thr Val Met Asn His Le - #u Gln Glu Thr Ala Leu    #80    - Asn His Val Lys Ser Ala Gly Leu Leu Gly As - #p Gly Phe Gly Ser Thr    #                95    - Pro Glu Met Cys Lys Lys Asn Leu Ile Trp Va - #l Val Thr Arg Met Gln    #           110    - Val Val Val Glu Arg Tyr Pro Thr Trp Gly As - #p Ile Val Gln Val Asp    #       125    - Thr Trp Val Ser Gly Ser Gly Lys Asn Gly Me - #t Arg Arg Asp Trp Leu    #   140    - Leu Arg Asp Ser Lys Thr Gly Glu Ile Leu Th - #r Arg Ala Ser Ser Val    145                 1 - #50                 1 - #55                 1 -    #60    - Trp Val Met Met Asn Lys Leu Thr Arg Arg Le - #u Ser Lys Ile Pro Glu    #               175    - Glu Val Arg Gln Glu Ile Gly Ser Tyr Phe Va - #l Asp Ser Asp Pro Ile    #           190    - Leu Glu Glu Asp Asn Arg Lys Leu Thr Lys Le - #u Asp Asp Asn Thr Ala    #           205    - Asp Tyr Ile Arg Thr Gly Leu Ser Pro Arg Tr - #p Ser Asp Leu Asp Ile    #   220    - Asn Gln His Val Asn Asn Val Lys Tyr Ile Gl - #y Trp Ile Leu Glu Ser    225                 2 - #30                 2 - #35                 2 -    #40    - Ala Pro Gln Pro Ile Leu Glu Ser His Glu Le - #u Ser Ser Met Thr Leu    #               255    - Glu Tyr Arg Arg Glu Cys Gly Arg Asp Ser Va - #l Leu Asp Ser Leu Thr    #           270    - Ala Val Ser Gly Ala Asp Met Gly Asn Leu Al - #a His Ser Gly His Val    #       285    - Glu Cys Lys His Leu Leu Arg Leu Glu Asn Gl - #y Ala Glu Ile Val Arg    #   300    - Gly Arg Thr Glu Trp Arg Pro Lys Pro Val As - #n Asn Phe Gly Val Val    305                 3 - #10                 3 - #15                 3 -    #20    - Asn Gln Val Pro Ala Glu Ser Thr                    325    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1174 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:    - ATAGGAGGTG GGAGAATGGG TATAGAATAA CATCAATGGC AGCAACTGCG GA - #TCAAGCAG      60    - CTTTCATATT AAGCATACCA AAGCGTAAGA TGGTGGATGA AACTCAAGAG AC - #TCTCCGCA     120    - CCACCGCCTT TCCAAGTACT CATGTCAAGG TTGGTTTCTT TAGCTTTGAA CA - #CAGATTTG     180    - GATCTTTTTG TTTTGTTTCC ATATACTTAG GACCTGAGAG CTTTTGGTTG AT - #TTTTTTTT     240    - CAGGACAAAT GGGCGAAGAA TCTGTACATT GCATCAATAT GCTATGGCAG GA - #CAGTGTGC     300    - TGATACACAC TTAAGCATCA TGTGGAAAGC CAAAGACAAT TGGAGCGAGA CT - #CAGGGTCG     360    - TCATAATACC AATCAAAGAC GTAAAACCAG ACGCAACCTC TTTGGTTGAA TG - #TAATGAAA     420    - GGGATGTGTC TTGGTATGTA TGTACGAATA ACAAAAGAGA AGATGGAATT AG - #TAGTAGAA     480    - AATATTTGGG AGCTTTTTAA GCCCTTCAAG TGTGCTTTTT ATCTTATTGA TA - #TCATCCAT     540    - TTGCGTTGTT TAATGCGTCT CTAGATATGT TCCTATATCT TTCTCAGTGT CT - #GATAAGTG     600    - AAATGTGAGA AAACCATACC AAACCAAAAT ATTCAAATCT TATTTTTAAT AA - #TGTTGAAT     660    - CACTCGGAGT TGCCACCTTC TGTGCCAATT GTGCTGAATC TATCACACTA GA - #AAAAAACA     720    - TTTCTTCAAG GTAATGACTT GTGGACTATG TTCTGAATTC TCATTAAGTT TT - #TATTTTCT     780    - GAAGTTTAAG TTTTTACCTT CTGTTTTGAA ATATATCGTT CATAAGATGT CA - #CGCCAGGA     840    - CATGAGCTAC ACATCGCACA TAGCATGCAG ATCAGGACGA TTTGTCACTC AC - #TTCAAACA     900    - CCTAAGAGCT TCTCTCTCAC AGCGCACACA CATATGCATG CAATATTTAC AC - #GTGATCGC     960    - CATGCAAATC TCCATTCTCA CCTATAAATT AGAGCCTCGG CTTCACTCTT TA - #CTCAAACC    1020    - AAAACTCATC ACTACAGAAC ATACACAAAT GGCGAACAAG CTCTTCCTCG TC - #TCGGCAAC    1080    - TCTCGCCTTG TTCTTCCTTC TCACCAATGC CTCCGTCTAC AGGACGGTTG TG - #GAAGTCGA    1140    #      1174        AATC CAGCCGGCCC ATTT    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1174 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:    - TATCCTCCAC CCTCTTACCC ATATCTTATT GTAGTTACCG TCGTTGACGC CT - #AGTTCGTC      60    - GAAAGTATAA TTCGTATGGT TTCGCATTCT ACCACCTACT TTGAGTTCTC TG - #AGAGGCGT     120    - GGTGGCGGAA AGGTTCATGA GTACAGTTCC AACCAAAGAA ATCGAAACTT GT - #GTCTAAAC     180    - CTAGAAAAAC AAAACAAAGG TATATGAATC CTGGACTCTC GAAAACCAAC TA - #AAAAAAAA     240    - GTCCTGTTTA CCCGCTTCTT AGACATGTAA CGTAGTTATA CGATACCGTC CT - #GTCACACG     300    - ACTATGTGTG AATTCGTAGT ACACCTTTCG GTTTCTGTTA ACCTCGCTCT GA - #GTCCCAGC     360    - AGTATTATGG TTAGTTTCTG CATTTTGGTC TGCGTTGGAG AAACCAACTT AC - #ATTACTTT     420    - CCCTACACAG AACCATACAT ACATGCTTAT TGTTTTCTCT TCTACCTTAA TC - #ATCATCTT     480    - TTATAAACCC TCGAAAAATT CGGGAAGTTC ACACGAAAAA TAGAATAACT AT - #AGTAGGTA     540    - AACGCAACAA ATTACGCAGA GATCTATACA AGGATATAGA AAGAGTCACA GA - #CTATTCAC     600    - TTTACACTCT TTTGGTATGG TTTGGTTTTA TAAGTTTAGA ATAAAAATTA TT - #ACAACTTA     660    - GTGAGCCTCA ACGGTGGAAG ACACGGTTAA CACGACTTAG ATAGTGTGAT CT - #TTTTTTGT     720    - AAAGAAGTTC CATTACTGAA CACCTGATAC AAGACTTAAG AGTAATTCAA AA - #ATAAAAGA     780    - CTTCAAATTC AAAAATGGAA GACAAAACTT TATATAGCAA GTATTCTACA GT - #GCGGTCCT     840    - GTACTCGATG TGTAGCGTGT ATCGTACGTC TAGTCCTGCT AAACAGTGAG TG - #AAGTTTGT     900    - GGATTCTCGA AGAGAGAGTG TCGCGTGTGT GTATACGTAC GTTATAAATG TG - #CACTAGCG     960    - GTACGTTTAG AGGTAAGAGT GGATATTTAA TCTCGGAGCC GAAGTGAGAA AT - #GAGTTTGG    1020    - TTTTGAGTAG TGATGTCTTG TATGTGTTTA CCGCTTGTTC GAGAAGGAGC AG - #AGCCGTTG    1080    - AGAGCGGAAC AAGAAGGAAG AGTGGTTACG GAGGCAGATG TCCTGCCAAC AC - #CTTCAGCT    1140    #      1174        TTAG GTCGGCCGGG TAAA    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1303 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #10:    - ACGCACTTAC CTAGAGCTTG CAACATCAGG CAAGTTAGCA TTTGCCCCTT CC - #AGAAGACC      60    - ATGCCTGGGC CCGGCTTCTA CTAGATTCCA AACGAATATC CTCGAGAGTG TG - #TATACCAC     120    - GGTGATATGA GTGTGGTTGT TGATGTATGT TAACACTACA TAGTCATGGT GT - #GTGTTCCA     180    - TAAATAATGT ACTAATGTAA TAAGAACTAC TCCGTAGACG GTAATAAAAG AG - #AAGTTTTT     240    - TTTTTTTACT CTTGCTACTT TCCTATAAAG TGATGATTAA CAACAGATAC AC - #CAAAAAGA     300    - AAACAATTAA TCTATATTCA CAATGAAGCA GTACTAGTCT ATTGAACATG TC - #AGATTTTC     360    - TTTTTCTAAA TGTCTAATTA AGCCTTCAAG GCTAGTGATG ATAAAAGATC AT - #CCAATGGG     420    - ATCCAACAAA GACTCAAATC TGGTTTTGAT CAGATACTTC AAAACTATTT TT - #GTATTCAT     480    - TAAATTATGC AAGTGTTCTT TTATTTGGTG AAGACTCTTT AGAAGCAAAG AA - #CGACAAGC     540    - AGTAATAAAA AAAACAAAGT TCAGTTTTAA GATTTGTTAT TGACTTATTG TC - #ATTTGAAA     600    - AATATAGTAT GATATTAATA TAGTTTTATT TATATAATGC TTGTCTATTC AA - #GATTTGAG     660    - AACATTAATA TGATACTGTC CACATATCCA ATATATTAAG TTTCATTTCT GT - #TCAAACAT     720    - ATGATAAGAT GGTCAAATGA TTATGAGTTT TGTTATTTAC CTGAAGAAAA GA - #TAAGTGAG     780    - CTTCGAGTTT CTGAAGGGTA CGTGATCTTC ATTTCTTGGC TAAAAGCGAA TA - #TGACATCA     840    - CCTAGAGAAA GCCGATAATA GTAAACTCTG TTCTTGGTTT TTGGTTTAAT CA - #AACCGAAC     900    - CGGTAGCTGA GTGTCAAGTC AGCAAACATC GCAAACCATA TGTCAATTCG TT - #AGATTCCC     960    - GGTTTAAGTT GTAAACCGGT ATTTCATTTG GTGAAAACCC TAGAAGCCAG CC - #ACCTTTTT    1020    - AATCTAATTT TTGCAAACGA GAAGTCACCA CACCTCTCCA CTAAAACCCT GA - #ACCTTACT    1080    - GAGAGAAGCA GAGCAAAAGA ACAAATAAAA CCCGAAGATG AGACCACCAC GT - #GCGGCGGG    1140    - ACGTTCAGGG GACGGGGAGG AAGAGAATGC GGCGGTTTGG TGGCGGCGGC GG - #ACGTTTGG    1200    - TGGCGGCGGT GGACGTTTTG GTGGCGGCGG TGGACCTTTG GTGGTGGATA TC - #GTGACGAA    1260    #                 130 - #3GTTCGTTT ACTCTTTTCT TAG    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1303 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #11:    - TGCGTGAATG GATCTCGAAC GTTGTAGTCC GTTCAATCGT AAACGGGGAA GG - #TCTTCTGG      60    - TACGGACCCG GGCCGAAGAT GATCTAAGGT TTGCTTATAG GAGCTCTCAC AC - #ATATGGTG     120    - CCACTATACT CACACCAACA ACTACATACA ATTGTGATGT ATCAGTACCA CA - #CACAAGGT     180    - ATTTATTACA TGATTACATT ATTCTTGATG AGGCATCTGC CATTATTTTC TC - #TTCAAAAA     240    - AAAAAAATGA GAACGATGAA AGGATATTTC ACTACTAATT GTTGTCTATG TG - #GTTTTTCT     300    - TTTGTTAATT AGATATAAGT GTTACTTCGT CATGATCAGA TAACTTGTAC AG - #TCTAAAAG     360    - AAAAAGATTT ACAGATTAAT TCGGAAGTTC CGATCACTAC TATTTTCTAG TA - #GGTTACCC     420    - TAGGTTGTTT CTGAGTTTAG ACCAAAACTA GTCTATGAAG TTTTGATAAA AA - #CATAAGTA     480    - ATTTAATACG TTCACAAGAA AATAAACCAC TTCTGAGAAA TCTTCGTTTC TT - #GCTGTTCG     540    - TCATTATTTT TTTTGTTTCA AGTCAAAATT CTAAACAATA ACTGAATAAC AG - #TAAACTTT     600    - TTATATCATA CTATAATTAT ATCAAAATAA ATATATTACG AACAGATAAG TT - #CTAAACTC     660    - TTGTAATTAT ACTATGACAG GTGTATAGGT TATATAATTC AAAGTAAAGA CA - #AGTTTGTA     720    - TACTATTCTA CCAGTTTACT AATACTCAAA ACAATAAATG GACTTCTTTT CT - #ATTCACTC     780    - GAAGCTCAAA GACTTCCCAT GCACTAGAAG TAAAGAACCG ATTTTCGCTT AT - #ACTGTAGT     840    - GGATCTCTTT CGGCTATTAT CATTTGAGAC AAGAACCAAA AACCAAATTA GT - #TTGGCTTG     900    - GCCATCGACT CACAGTTCAG TCGTTTGTAG CGTTTGGTAT ACAGTTAAGC AA - #TCTAAGGG     960    - CCAAATTCAA CATTTGGCCA TAAAGTAAAC CACTTTTGGG ATCTTCGGTC GG - #TGGAAAAA    1020    - TTAGATTAAA AACGTTTGCT CTTCAGTGGT GTGGAGAGGT GATTTTGGGA CT - #TGGAATGA    1080    - CTCTCTTCGT CTCGTTTTCT TGTTTATTTT GGGCTTCTAC TCTGGTGGTG CA - #CGCCGCCC    1140    - TGCAAGTCCC CTGCCCCTCC TTCTCTTACG CCGCCAAACC ACCGCCGCCG CC - #TGCAAACC    1200    - ACCGCCGCCA CCTGCAAAAC CACCGCCGCC ACCTGGAAAC CACCACCTAT AG - #CACTGCTT    1260    #                 130 - #3CAAGCAAA TGAGAAAAGA ATC    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  36 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #12:    #       36         GCGA TTTACTGCTG CTTTTC    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #13:    #                24ACTG CGGA    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  23 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #14:    #                23GCAT CAT    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  34 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #15:    #        34        GTAT GTTCTGTAGT GATG    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  35 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #16:    #       35         GCGG ATCAAGCAGC TTTCA    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  34 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #17:    #        34        GATT CCAAACGAAA TCCT    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #18:    #                24GCAT TTGC    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  34 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #19:    #        34        GGTC CTTCGTCAGC ATAT    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  24 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #20:    #                24CACT GGGA    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  36 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #21:    #       36         ACGA GGTCCTTCGT CACGAT    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  36 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #22:    #       36         CTGG TACCTAGATT CCAAAC    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  20 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #23:    # 20               CTCA    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  39 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #24:    #    39            GCTA AAGAGTGAAG CCGAGGCTC    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  10 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #25:    #        10    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  12 base - # pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #26:    #       12    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1688 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  YES    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #27:    - TTTTTTTTTT TTTTAATTAC AATGAGAATG AGATTTACTG CTGCTTTTCC CC - #CTTACCCA      60    - CCAAAGTATC ACAAATTAGA AGGTGTATAT ATTTAAAGAC ATGTAGATTG AT - #TGGATAGA     120    - CACAAGCAAA ACTCTCCCCA GGGAACCAGA ATAAGTCTAA ACATAGCAAG GA - #GACTGATG     180    - CAACTCCAAT CGTTAACCAT TTCAAATCTT AGGTGCTTTC TGCTGGAACC TG - #GTTCACAA     240    - CACCAAAGTT GTTCACAGGT TTGGGCCTCC ACTCAGTCCT GCCCCTCACA AT - #CTCAGCAC     300    - CATTTTCCAG TCGAAGCAAA TGCTTGCACT CAACATGCCC GCTGTGAGCT AG - #ATTGCCCA     360    - TGTCGGCCCC AGATACAGCA GTCAGGGAAT CCAGCACACT GTCCCTACCA CA - #CTCTCTCC     420    - TATACTCTAA AGTCATGGAA GAAAGCTCAT GACTCTCCAA GATTGGCTGT GG - #AGCACTCT     480    - CCAGAATCCA GCCAATGTAC TTCACATTGT TGACATGCTG ATTGATATCT AG - #ATCACTCC     540    - ACCTAGGACT TAAACCGGTA CGAATATAAT CCGCTGTGTT GTCGTCAAGT TT - #AGTCAGTT     600    - TTCTGTTATC CTCTTCCAGA ATTGGATCAG AATCCACAAA ATAAGATCCT AT - #CTCCTGTC     660    - TGACTTCTTC TGGAATTTTA GACAGCCTCC GTGTTAGCTT ATTCATCATG AC - #CCAAACAC     720    - TGGAAGCTCT TGTCAAGATT TCACCAGTTT TGGAGTCACG TAAAAGCCAA TC - #ACGACGCA     780    - TACCATTCTT CCCTGATCCA GAAACCCAAG TGTCCACTTG AACTATGTCA CC - #CCATGTAG     840    - GATAGCGTTC CACCACAACC TGCATCCGAG TAACCACCCA TATCAAGTTC TT - #TTTGCACA     900    - TTTCTGGCGT GGAACCAAAG CCATCACCAA GAAGCCCAGC ACTTTTAACA TG - #ATTAAGTG     960    - CAGTTTCTTG CAAATGGTTC ATTACTGTTT CTATAGATGC GGTACGATCA GC - #ACCAATCT    1020    - CATATGATCT AATAGAAAAG TTTTCACGGA ACACAAGACC ATCCTGAACA AT - #TTTTCCTA    1080    - TCCCAAAGGG GTCAATAAGC ATGTCAGGTC GCCGTGGCTT CCAATCAAGC AT - #CATCCACT    1140    - GCTTTTCAGC GGCCAAGAAA ATTGTTGTGA TAGCAGCAAG AAGCATGCTC CA - #ATCAGGCA    1200    - ACTGGTTGAT AAAAGTTCTG GGGGGAGGCG AAGGTAGATC ATCATCATGC TT - #GAAGCTTT    1260    - CTTTAGATGT AACAACTGTG GTTCCATTAA TTTTCGAAGG GGCTTGCGCC TT - #TGCCTTCA    1320    - AGCCACCAGA AGACGCAGAT TTGGATTTTA GTCCTCCAAG GTTTGCAGGC CC - #ACCACCAA    1380    - GTTTGCTGCC TGCTCCACCA GAGTCCGGCG AGGGTGAAGT AACAGGGAAA AA - #TGATGAAG    1440    - TAGCAGCTGT TGCCACCATA ATGAATTTCT AAGGTCGCTT CTCCGGTAGA AC - #GTCTAGTC    1500    - TAGAAATGCA GAAAAAGCGG GTTTGGTCTT GTGTTATCTG AGGAGTTGGA TC - #TGACTGAG    1560    - TGAAGAAGAA GAAGAAGATG GAGAGAGAGA GAAGGAGAAA AGCTGGAAAC AG - #GGAAGAAG    1620    - GGCTATTGTG TCATTTTGCG TCCTTGTTGT TTCCCTAATT TTGGAAAAGA GA - #GAGACAGT    1680    #        1688    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1483 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (iv) ANTI-SENSE:  YES    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #28:    - TTTTTTTTTT TTTTTTTTTA AACCCCCAAA ATAAAATACA TTTAAATAAT AT - #TAGGATAA      60    - GAAAGTTATT TGCCTTTGTC TGGCACCCAA AAGAAAAAAT AAATATAATA AA - #AAGGGACC     120    - TCCAAGAAGA AAAAAAATAA GAACCAAAGA AAATATACAA AGGTGGCCCA AA - #CTGTTTTT     180    - GAGTAGTGGT GGTTGCAAGC AAGGTAGTAT AGTTTTACAA ACGAACCAAA GA - #ACCCATGT     240    - TTGCTATATT CTTTTCACGG TGTGATGTCC CAAGTTGTTG TTGATGTTTT GG - #AACTCCAC     300    - TCTGTTCTTC CTCTCACCAC TTCAGCTCCA TCCTGGAGAC GGAGCAGATG CT - #GACATTCC     360    - ACTTCACCAG CCGTCCCGAG GCTACCGATA TCGCAGCCCG AAACCGCGGT GA - #GGGACTGA     420    - AGCACACTGT CCCTCCCGCA CTCCCTGCGA TACTCCAGAG TCATGCTTTT CA - #GCTTCTGA     480    - CTCTCCATCA TCCCCACAGG TGCACTCTCC AGTATCCACC CGATGTACTT CA - #CATTGTTA     540    - ACGTGCTGGT TAACATCCAA GTCACTCCAA CGCGGAGTGA GACCAGAACG AA - #CATAGTCA     600    - GCAGTCTTGT CATCAAGTTT TGTTAACTTT CTGCTGTCCT CGGCAAGGAC TG - #GGTCAGAA     660    - TTAACAAAGT AAGGCTCTAT CTCCCCTCGA ACCTCTTCAG GAATCTTTGA TA - #ATCTTCTT     720    - GTCAGTTTAT TCATCATCAC CCACACACTT GATGCTCTTG TTAAAATTTC TC - #CAGTATTG     780    - CCATCTCGAA CTAGCCAATC ACGACGCATA CCGTTCTTTC CAGACTGGCT CA - #CCCATGTA     840    - TCTACTTCCA CAACATCTCC CCAAGTAGGA TATTTATCAA CGACAACCTG CA - #TACGAGTA     900    - ACAACCCAAA TCAAGTTCTT CTTAACCATC TCAGGAGTAG AACCAAACCC AT - #CTCCAAGC     960    - AGTCCAGCAG TCTTAACATG GTTTAGTGCC GTTTCCTGTA AATGATTCAT AA - #CCGTTTCT    1020    - ATAGACGCAG AGCGATCAGC ACCTATCTCA TAAGACCGAA TAGAGAAATT CT - #GACGGAAC    1080    - ACAAGCCCAT CCTGAACGAT CCTCCCTAAC CCAAACGGAT CCATAATCAC GT - #CAGAGCGC    1140    - CTCGGTTTCC AGTCAAGCAT CATCCACTGC TTCTCAGCCG CCAAGAAGAC GG - #TTGTTATT    1200    - GCAGCAAGAA GCATGCTCCA GTCAGGCAGC TGGTTGATGA ACGTCCTCGG TG - #CTGCGGGA    1260    - TGCTGTGAGG ACGTCTCGTT ATCAGGCTTC ACCGAGCCAG AAGGGAGACC GA - #CTCTCTTG    1320    - CCGTTGATCT TGGGTGGGGC CTGAGCGTTT GGTTTAACCT TCATCTGCCG GA - #GGAGTTTG    1380    - GAGTGGGGAA GATGCCGGAG AAGTTGGTGG AGGTGGTGAC TTTGTTGGTT TT - #TGCGGTGG    1440    #                 148 - #3AGGGAAGA ATGAGCTCGT GCC    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  324 ami - #no acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  unkn - #own              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE:  protein    -    (iii) HYPOTHETICAL:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #29:    - Leu Pro Asp Trp Ser Met Leu Leu Ala Ala Il - #e Thr Thr Val Phe Leu    #                15    - Ala Ala Glu Lys Gln Trp Met Met Leu Asp Tr - #p Lys Pro Arg Arg Ser    #            30    - Asp Val Ile Met Asp Pro Phe Gly Leu Gly Ar - #g Ile Val Gln Asp Gly    #        45    - Leu Val Phe Arg Gln Asn Phe Ser Ile Arg Se - #r Tyr Glu Ile Gly Ala    #    60    - Asp Arg Ser Ala Ser Ile Glu Thr Val Met As - #n His Leu Gln Glu Thr    #80    - Ala Leu Asn His Val Lys Thr Ala Gly Leu Le - #u Gly Asp Gly Phe Gly    #                95    - Ser Thr Pro Glu Met Val Lys Lys Asn Leu Il - #e Trp Val Val Thr Arg    #           110    - Met Gln Val Val Val Asp Lys Tyr Pro Thr Tr - #p Gly Asp Val Val Glu    #       125    - Val Asp Thr Trp Val Ser Gln Ser Gly Lys As - #n Gly Met Arg Arg Asp    #   140    - Trp Leu Val Arg Asp Gly Asn Thr Gly Glu Il - #e Leu Thr Arg Ala Ser    145                 1 - #50                 1 - #55                 1 -    #60    - Ser Val Trp Val Met Met Asn Lys Leu Thr Ar - #g Arg Leu Ser Lys Ile    #               175    - Pro Glu Glu Val Arg Gly Glu Ile Glu Pro Ty - #r Phe Val Asn Ser Asp    #           190    - Pro Val Leu Ala Glu Asp Ser Arg Lys Leu Th - #r Lys Leu Asp Asp Lys    #       205    - Thr Ala Asp Tyr Val Arg Ser Gly Leu Thr Pr - #o Arg Trp Ser Asp Leu    #   220    - Asp Val Asn Gln His Val Asn Asn Val Lys Ty - #r Ile Gly Trp Ile Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Glu Ser Ala Pro Val Gly Met Met Glu Ser Gl - #n Lys Leu Lys Ser Met    #               255    - Thr Leu Glu Tyr Arg Arg Glu Cys Gly Arg As - #p Ser Val Leu Gln Ser    #           270    - Leu Thr Ala Val Ser Gly Cys Asp Ile Gly Se - #r Leu Gly Thr Ala Gly    #       285    - Glu Val Glu Cys Gln His Leu Leu Arg Leu Gl - #n Asp Gly Ala Glu Val    #   300    - Val Arg Gly Arg Thr Glu Trp Ser Ser Lys Th - #r Ser Thr Thr Thr Trp    305                 3 - #10                 3 - #15                 3 -    #20    - Asp Ile Thr Pro    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  324 ami - #no acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  unkn - #own              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE:  protein    -    (iii) HYPOTHETICAL:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #30:    - Leu Leu Ala Ala Ile Thr Thr Ile Phe Leu Al - #a Ala Glu Lys Gln Trp    #                15    - Met Met Leu Asp Trp Lys Pro Arg Arg Pro As - #p Met Leu Ile Asp Pro    #            30    - Phe Gly Ile Gly Lys Ile Val Gln Asp Gly Le - #u Val Phe Arg Glu Asn    #        45    - Phe Ser Ile Arg Ser Tyr Glu Ile Gly Ala As - #p Arg Thr Ala Ser Ile    #    60    - Glu Thr Val Met Asn His Leu Gln Glu Thr Al - #a Leu Asn His Val Lys    #80    - Ser Ala Gly Leu Leu Gly Asp Gly Phe Gly Se - #r Thr Pro Glu Met Cys    #                95    - Lys Lys Asn Leu Ile Trp Val Val Thr Arg Me - #t Gln Val Val Val Glu    #           110    - Arg Tyr Pro Thr Trp Gly Asp Ile Val Gln Va - #l Asp Thr Trp Val Ser    #       125    - Gly Ser Gly Lys Asn Gly Met Arg Arg Asp Tr - #p Leu Leu Arg Asp Ser    #   140    - Lys Thr Gly Glu Ile Leu Thr Arg Ala Ser Se - #r Val Trp Val Met Met    145                 1 - #50                 1 - #55                 1 -    #60    - Asn Lys Leu Thr Arg Arg Leu Ser Lys Ile Pr - #o Glu Glu Val Arg Gln    #               175    - Glu Ile Gly Ser Tyr Phe Val Asp Ser Asp Pr - #o Ile Leu Glu Glu Asp    #           190    - Asn Arg Lys Leu Thr Lys Leu Asp Asp Asn Th - #r Ala Asp Tyr Ile Arg    #       205    - Thr Gly Leu Ser Pro Arg Trp Ser Asp Leu As - #p Ile Asn Gln His Val    #   220    - Asn Asn Val Lys Tyr Ile Gly Trp Ile Leu Gl - #u Ser Ala Pro Gln Pro    225                 2 - #30                 2 - #35                 2 -    #40    - Ile Leu Glu Ser His Glu Leu Ser Ser Met Th - #r Leu Glu Tyr Arg Arg    #               255    - Glu Cys Gly Arg Asp Ser Val Leu Asp Ser Le - #u Thr Ala Val Ser Gly    #           270    - Ala Asp Met Gly Asn Leu Ala His Ser Gly Hi - #s Val Glu Cys Lys His    #       285    - Leu Leu Arg Leu Glu Asn Gly Ala Glu Ile Va - #l Arg Gly Arg Thr Glu    #   300    - Trp Arg Pro Lys Pro Val Asn Asn Phe Gly Va - #l Val Asn Gln Val Pro    305                 3 - #10                 3 - #15                 3 -    #20    - Ala Glu Ser Thr    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1674 ba - #se pairs              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  linear    -     (ii) MOLECULE TYPE:  DNA (genomic)    -    (iii) HYPOTHETICAL:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #31:    - GCACGAGCTC GTGCCGAATT CGGCACGAGC GGCACGAGGA AAATACAGAG AG - #ACAAATTT      60    - AAAACAAAAC GAAAGGAGAT CGAGAGAGGA GAGAGGCGCA CACACACACA CA - #CAAAGGAG     120    - AACTTTAGGG TTTGGGGAGA CTCCGAAGAG ATTGGCGTAA CACTTCTGTC TT - #TGAACGCT     180    - TATCTTCCTC GTCATGGTGG CTACTTGCGC TACGTCGTCG TTTTTTCATG TT - #CCATCTTC     240    - TTCCTCGCTT GATACGAATG GGAAGGGGAA CAGAGTTGGG TCCACTAATT TT - #GCTGGACT     300    - TAACTCAACG CCAAGCTCTG GGAGGATGAA GGTTAAGCCA AACGCTCAGG CT - #CCACCCAA     360    - GATCAACGGG AAGAAAGCTA ACTTGCCTGG CTCTGTAGAG ATATCAAAGG CT - #GACAACGA     420    - GACTTCGCAG CCCGCACACG CACCGAGGAC GTTTATCAAC CAGCTGCCTG AC - #TGGAGTAT     480    - GCTGCTTGCT GCTATAACTA CCATTTTCTT GGCAGCGGAG AAACAGTGGA TG - #ATGCTTGA     540    - CTGGAAACCG AGGCGTTCTG ATATGATTAT GGATCCTTTT GGTTTAGGGA GA - #ATTGTTCA     600    - GGATGGTCTT GTGTTCCGTC AGAATTTTTC CATTAGGTCT TATGAAATAG GT - #GCTGATCG     660    - CTCTGCGTCT ATAGAAACTG TCATGAATCA TTTACAGGAA ACGGCGCTTA AT - #CATGTGAA     720    - GTCTGCCGGA CTGCTGGAAA ATGGGTTTGG GTCCACTCCT GAGATGTTTA AG - #AAGAATTT     780    - GATATGGGTC GTTGCTCGTA TGCAGGTTGT CGTTGATAAA TATCCTACTT GG - #GGAGATGT     840    - TGTGGAAGTG GATACTTGGG TTAGTCAGTC TGGAAAGAAT GGTATGCGTC GT - #GATTGGCT     900    - AGTTCGGGAT TGCAATACTG GAGAAATTGT AACGCGAGCA TCAAGTTTGT GG - #GTGATGAT     960    - GAATAAACTC ACAAGGAGAT TGTCAAAGAT TCCTGAAGAG GTTCGAGGGG AA - #ATAGAGCC    1020    - TTATTTTGTG AACTCTGATC CTGTCATTGC CGAAGACAGC AGAAAGTTAA CA - #AAACTTGA    1080    - TGACAAGACT GCTGACTATG TTCGTTCTGG TCTCACTCCG AGGTGGAGTG AC - #TTGGATGT    1140    - TAACCAGCAT GTTAACAATG TAAAGTACAT TGGGTGGATA CTGGAGAGTG CT - #CCAGCAGG    1200    - GATGCTGGAG AGTCAGAAGC TGAAAAGCAT GACTCTGGAG TATCGCAGGG AG - #TGCGGGAG    1260    - AGACAGTGTG CTTCAGTCTC TCACCGCAGT CTCTGGATGT GATGTCGGTA AC - #CTCGGGAC    1320    - AGCCGGGGAA GTGGAGTGTC AGCATTTGCT TCGACTCCAG GATGGAGCTG AA - #GTGGTGAG    1380    - AGGAAGAACA GAGTGGAGCT CCAAGACAGG AGCAACAACT TGGGACACTA CT - #ACATCGTA    1440    - AACATTGGTC CTTTGGTTCC TTTGTAAAAC TGTACCTGCT GCTACCTTCT TG - #CAACCACC    1500    - ACCTTTGTAT ATTTCTTCTT TTTTGTTTTT TATTTTGCTT CAATGGAGAT AT - #ATTATTAT    1560    - TTATTTAATC TTTCTATTTT TTTTGTTTTC TTATGGGAAA TGGGTGTATT AT - #GTGATATA    1620    - TTATTGTAAC CCCATGTGCC AGGGCAAGGC AATAACTTTC TTATCAAAAA AA - #AA    1674    - (2) INFORMATION FOR SEQ ID NO: 32:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  415 ami - #no acids              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  unkn - #own              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE:  protein    -    (iii) HYPOTHETICAL:  NO    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 32:    - Met Val Ala Thr Cys Ala Thr Ser Ser Phe Ph - #e His Val Pro Ser Ser    #                15    - Ser Ser Leu Asp Thr Asn Gly Lys Gly Asn Ar - #g Val Gly Ser Thr Asn    #            30    - Phe Ala Gly Leu Asn Ser Thr Pro Ser Ser Gl - #y Arg Met Lys Val Lys    #        45    - Pro Asn Ala Gln Ala Pro Pro Lys Ile Asn Gl - #y Lys Lys Ala Asn Leu    #    60    - Pro Gly Ser Val Glu Ile Ser Lys Ala Asp As - #n Glu Thr Ser Gln Pro    #80    - Ala His Ala Pro Arg Thr Phe Ile Asn Gln Le - #u Pro Asp Trp Ser Met    #                95    - Leu Leu Ala Ala Ile Thr Thr Ile Phe Leu Al - #a Ala Glu Lys Gln Trp    #           110    - Met Met Leu Asp Trp Lys Pro Arg Arg Ser As - #p Met Ile Met Asp Pro    #       125    - Phe Gly Leu Gly Arg Ile Val Gln Asp Gly Le - #u Val Phe Arg Gln Asn    #   140    - Phe Ser Ile Arg Ser Tyr Glu Ile Gly Ala As - #p Arg Ser Ala Ser Ile    145                 1 - #50                 1 - #55                 1 -    #60    - Glu Thr Val Met Asn His Leu Gln Glu Thr Al - #a Leu Asn His Val Lys    #               175    - Ser Ala Gly Leu Leu Glu Asn Gly Phe Gly Se - #r Thr Pro Glu Met Phe    #           190    - Lys Lys Asn Leu Ile Trp Val Val Ala Arg Me - #t Gln Val Val Val Asp    #       205    - Lys Tyr Pro Thr Trp Gly Asp Val Val Glu Va - #l Asp Thr Trp Val Ser    #   220    - Gln Ser Gly Lys Asn Gly Met Arg Arg Asp Tr - #p Leu Val Arg Asp Cys    225                 2 - #30                 2 - #35                 2 -    #40    - Asn Thr Gly Glu Ile Val Thr Arg Ala Ser Se - #r Leu Trp Val Met Met    #               255    - Asn Lys Leu Thr Arg Arg Leu Ser Lys Ile Pr - #o Glu Glu Val Arg Gly    #           270    - Glu Ile Glu Pro Tyr Phe Val Asn Ser Asp Pr - #o Val Ile Ala Glu Asp    #       285    - Ser Arg Lys Leu Thr Lys Leu Asp Asp Lys Th - #r Ala Asp Tyr Val Arg    #   300    - Ser Gly Leu Thr Pro Arg Trp Ser Asp Leu As - #p Val Asn Gln His Val    305                 3 - #10                 3 - #15                 3 -    #20    - Asn Asn Val Lys Tyr Ile Gly Trp Ile Leu Gl - #u Ser Ala Pro Ala Gly    #               335    - Met Leu Glu Ser Gln Lys Leu Lys Ser Met Th - #r Leu Glu Tyr Arg Arg    #           350    - Glu Cys Gly Arg Asp Ser Val Leu Gln Ser Le - #u Thr Ala Val Ser Gly    #       365    - Cys Asp Val Gly Asn Leu Gly Thr Ala Gly Gl - #u Val Glu Cys Gln His    #   380    - Leu Leu Arg Leu Gln Asp Gly Ala Glu Val Va - #l Arg Gly Arg Thr Glu    385                 3 - #90                 3 - #95                 4 -    #00    - Trp Ser Ser Lys Thr Gly Ala Thr Thr Trp As - #p Thr Thr Thr Ser    #               415    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid fragment encoding aplant acyl-ACP thioesterase wherein said thioesterase has a preferenceof at least two-fold for palmitoyl-ACP over either stearoyl-ACP oroleoyl-ACP and further wherein said isolated nucleic acid fragmenthybridizes to one of the nucleotide sequences set forth in SEQ ID NOS:1,2, and 31 under the following set of conditions: hybridization at 60° C.in 6×SCC, 0.1% SDS for 18 hr, washing at 60° C. in 0.2×SSC, 0.1% SDStwice for 10 min each.
 2. An isolated nucleic acid fragment encoding thesoybean seed acyl-ACP thioesterase encoded by nucleotides 1 to 1688 ofSEQ ID NO:1.
 3. An isolated nucleic acid fragment encoding the canolaseed acyl-ACP thioesterase encoded by nucleotides 1 to 1483 of SEQ IDNO:2.
 4. An isolated nucleic acid fragment encoding the canola seedacyl-ACP thioesterase encoded by nucleotides 1 to 1674 of SEQ ID NO:31.5. An isolated nucleic acid fragment encoding a plant acyl-ACPthioesterase wherein said thioesterase has a preference of at leasttwo-fold for palmitoyl-ACP over either stearoyl-ACP or oleoyl-ACP andfurther wherein said isolated nucleic acid fragment hybridizes tonucleotides 506 to 1477 of SEQ ID NO:1, nucleotides 255 to 1226 of SEQID NO:2, or nucleotides 479 to 1438 of SEQ ID NO:31 under the followingset of conditions: hybridization at 60° C. in 6×SCC, 0.1% SDS for 18 hr,washing at 60° C. in 0.2×SSC, 0.1% SDS twice for 10 min each.
 6. Theisolated nucleic acid fragment of claim 2 wherein the said nucleotidesequence encodes the catalytically active soybean seed palmitoyl-ACPthioesterase enzyme encoded by nucleotides 506 to 1477 of SEQ ID NO:1.7. The isolated nucleic acid fragment of claim 3 wherein the saidnucleotide sequence encodes the catalytically active canola seedpalmitoyl-ACP thioesterase enzyme encoded by nucleotides 255 to 1226 ofSEQ ID NO:2.
 8. The isolated nucleic acid fragment of claim 4 whereinthe said nucleotide sequence encodes the catalytically active canolaseed palmitoyl-ACP thioesterase enzyme encoded by nucleotides 479 to1438 of SEQ ID NO:31.
 9. An isolated nucleic-acid fragment encoding asoybean acyl-ACP thioesterase having the amino acid sequence of SEQ IDNO:29.
 10. An isolated nucleic-acid fragment encoding a rapeseedacyl-ACP thioesterase having the amino acid sequence of SEQ ID NO:30.11. An isolated nucleic-acid fragment encoding a rapeseed acyl-ACPthioesterase having the amino acid sequence of SEQ ID NO:32.
 12. Achimeric gene for transforming a plant of an oil producing speciescomprising the nucleic acid fragment of claim 1 operably linked inantisense orientation to regulatory sequences, wherein said chimericgene causes inhibition of expression of palmitoyl-ACP thioesterase inseed of said plant wherein said inhibition results in lower-than-normallevels of saturated fatty acids.
 13. A chimeric gene for transforming aplant of an oil producing species comprising the nucleic acid fragmentof claim 1 operably linked in sense orientation to regulatory sequences,wherein said chimeric gene causes sense elevation or co-suppression ofpalmitoyl-ACP thioesterase in seed of said plant.
 14. A chimeric genefor transforming a plant of an oil producing species comprising thenucleic acid fragment of claim 2 operably linked in antisenseorientation to regulatory sequences, wherein said chimeric gene causesinhibition of expression of palmitoyl-ACP thioesterase in seed of saidplant.
 15. A chimeric gene for transforming a plant of an oil producingspecies comprising the nucleic acid fragment of claim 2 operably linkedin sense orientation to regulatory sequences, wherein said chimeric genecauses sense elevation or co-suppression of palmitoyl-ACP thioesterasein seed of said plant.
 16. A chimeric gene for transforming a plant ofan oil producing species comprising the nucleic acid fragment of claim 3or 4 operably linked in antisense orientation to regulatory sequences,wherein said chimeric gene causes inhibition of expression ofpalmitoyl-ACP thioesterase in seed of said plant.
 17. A chimeric genefor transforming a plant of an oil producing species comprising thenucleic acid fragment of claim 3 or 4 operably linked in senseorientation to regulatory sequences, wherein said chimeric gene causeselevation or co-suppression of palmitoyl-ACP thioesterase in seed ofsaid plant.
 18. The chimeric gene of claim 12 wherein said plant of anoil producing species is selected from the group consisting of soybean,rapeseed, sunflower, cotton, cocoa, peanut, safflower, and corn.
 19. Thechimeric gene of claim 13 wherein said plant of an oil producing speciesis selected from the group consisting of soybean, rapeseed, sunflower,cotton, cocoa, peanut, safflower, and corn.
 20. A plant cell transformedwith the chimeric gene of claim
 12. 21. A plant cell transformed withthe chimeric gene of claim
 13. 22. The plant cell, as described in claim20, wherein the plant cell is selected from the group consisting ofsoybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, andcorn.
 23. The plant cell, as described in claim 21, wherein the plantcell is selected from the group consisting of soybean, rapeseed,sunflower, cotton, cocoa, peanut, safflower, and corn.
 24. A method ofproducing plant seed oil comprising lower-than-normal levels of palmiticand stearic acids comprising:(a) transforming plant cells of an oilproducing species with the chimeric gene of claim 12 or the chimericgene of claim 13, (b) growing fertile plants from the transformed plantcells obtained from step (a), (c) screening progeny seeds from saidfertile plants for lower-than-normal levels palmitic and stearic acids,and (d) crushing said progeny seeds to obtain said plant seed oilcomprising lower-than-normal levels of palmitic and stearic acids.
 25. Amethod of producing plant seed oil comprising higher-than-normal levelsof palmitic and stearic acids comprising:(a) transforming plant cells ofan oil producing species with the chimeric gene of claim 13, (b) growingfertile plants from the transformed plant cells obtained from step (a),(c) screening progeny seeds from said fertile plants forhigher-than-normal levels of palmitic and stearic acids, and (d)crushing said progeny seeds to obtain said plant seed oil comprisinghigher-than-normal levels of palmitic and stearic acids.
 26. A method ofproducing soybean seed oil comprising lower-than-normal levels ofpalmitic and stearic acids comprising:(a) transforming soybean cellswith the chimeric gene of claim 14, (b) growing fertile soybean plantsfrom the transformed soybean cells obtained from step (a), (c) screeningprogeny seeds from said fertile soybean plants for lower-than-normallevels of palmitic and stearic acids, and (d) crushing said progenyseeds to obtain said soybean seed oil comprising lower-than-normallevels of palmitic and stearic acids.
 27. A method of producing soybeanseed oil comprising higher-than-normal levels of palmitic and stearicacids comprising:(a) transforming soybean cells with the chimeric geneof claim 15, (b) growing fertile soybean plants from the transformedsoybean cells obtained from step (a), (c) screening progeny seeds fromsaid fertile soybean plants for higher-than-normal levels of palmiticand stearic acids, and (d) crushing said progeny seeds to obtain saidsoybean seed oil comprising higher-than-normal levels of palmitic andstearic acids.
 28. A method of producing rapeseed seed oil comprisinglower-than-normal levels of palmitic and stearic acids comprising:(a)transforming rapeseed cells with the chimeric gene of claim 16, (b)growing fertile rapeseed plants from the transformed rapeseed cellsobtained from step (a), (c) screening progeny seeds from said fertilerapeseed plants for lower-than-normal levels of palmitic and stearicacids, and (d) crushing said progeny seeds to obtain said rapeseed seedoil comprising lower-than-normal levels of palmitic and stearic acids.29. A method of producing rapeseed seed oil comprisinghigher-than-normal levels of palmitic and stearic acids comprising:(a)transforming rapeseed cells with the chimeric gene of claim 17, (b)growing fertile rapeseed plants from the transformed rapeseed cellsobtained from step (a), (c) screening progeny seeds from said fertilerapeseed plants for higher-than-normal levels of palmitic and stearicacids, and (d) crushing said progeny seeds to obtain said rapeseed seedoil comprising higher-than-normal levels of palmitic and stearic acids.30. The method of claim 24 wherein the plant cells are from an oilproducing species selected from the group consisting of soybean,rapeseed, sunflower, cotton, cocoa, peanut, safflower, and corn.
 31. Themethod of claim 25 wherein the plant cells are from an oil producingspecies selected from the group consisting of soybean, rapeseed,sunflower, cotton, cocoa, peanut, safflower, and corn.
 32. The method ofclaim 24 wherein the transforming of step (a) is accomplished by aprocess selected from the group consisting of Agrobacterium infection,electroporation, and high-velocity ballistic bombardment.
 33. The methodof claim 25 wherein the transforming of step (a) is accomplished by aprocess selected from the group consisting of Agrobacterium infection,electroporation, and high-velocity ballistic bombardment.